CN110577599A

CN110577599A - humanized anti-programmed death-1 (PD-1) antibody and application thereof

Info

Publication number: CN110577599A
Application number: CN201910394015.3A
Authority: CN
Inventors: 马冬磊; 林科佳; 魏宗科; 胡锐
Original assignee: Shenzhen Run Ke Biotechnology Co Ltd
Current assignee: Shenzhen Run Ke Biotechnology Co Ltd
Priority date: 2019-05-13
Filing date: 2019-05-13
Publication date: 2019-12-17

Abstract

The invention discloses a humanized anti-programmed death-1 (PD-1) antibody or antigen binding fragment, which comprises a heavy chain variable region, wherein the heavy chain variable region comprises at least one amino acid sequence shown as SEQ ID NO 1-2, SEQ ID NO 3-5, SEQ ID NO 6-8, SEQ ID NO 9-11 and SEQ ID NO 12. The antibody or the antigen binding fragment has bioactivity of inducing interleukin-2 release in a mixed lymphocyte experiment, can be combined with a human-derived programmed death-1 (PD-1) protein target with high affinity, can be widely applied to in vitro detection of PD-1 protein, and can be used for preparing a medicament for enhancing T cell immune response and treating autoimmune diseases, inflammatory diseases, allergic reactions, graft rejection, cancers or immune system disorder medicaments.

Description

humanized anti-programmed death-1 (PD-1) antibody and application thereof

Technical Field

the invention belongs to the technical field of molecular immunology, and particularly relates to an anti-programmed death-1 (PD-1) antibody or a functional fragment thereof and application thereof in medicines for treating diseases such as tumors and the like.

background

The adaptive immune response in the body involves the activation, selection and clonal proliferation of two major classes of lymphocytes, namely T cells and B cells. After the body encounters a foreign antigen, its T cells begin to proliferate and differentiate into antigen-specific responder cells, while the B cells also proliferate and differentiate to form antibody-secreting cells. T cell activation is a multi-step process that requires the transduction of back and forth signals between T cells and Antigen Presenting Cells (APCs). For activation of T cells, two types of signals must be transmitted to other T cells: the first class of costimulatory signaling is regulated by antigen-specific T cell receptors (TcR), which in turn manifest as specific immune responses of the body; while a second class of co-stimulatory signaling regulates the intensity of specific immune responses, the signal of which is transmitted through helper receptors on T cells.

The first class of costimulatory signals, among others, by first activating the CD28 receptor, which then binds to ligand B7-1 or B7-2. In contrast, cytotoxic T lymphocyte antigen 4(CTLA-4) receptors also attenuate T cell immune responses by binding to the same ligand, B7-1 or B7-2, so that CTLA-4 signaling acts as a co-stimulatory signaling mediated by CD28, and at high antigen concentrations, co-stimulatory signaling mediated by CD28 overrides the inhibitory effect of CTLA-4 signaling. Transient regulation of CD28 and CTLA-4 expression maintains a balance of activation and inhibition signaling, i.e., protects the body from autoimmunity while ensuring that an effective immune response is developed.

recently, researchers have identified molecular homology of CD28, CTLA-4, and their class B7 ligands. Induced T-cell COStimulator (ICOS), also called CD278 is a costimulatory signal receptor similar to CD28, Programmed death-1 (PD-1) is PD-1 or CD279 is an inhibitory receptor that acts in parallel with CTLA-4. PD-1 is a 50-55 Dalton size type I transmembrane receptor that was originally identified when T lymphocytes were studied for their induction of activated apoptosis. PD-1 is expressed in activated T cells, B cells, and macrophages. The ligands are PD-L1(B7H1/CD274) and PD-L2(B7-DC/CD273), which are members of the B7 family.

PD-1 is also a member of the immunoglobulin (Ig) superfamily, which comprises a single domain such as the immunoglobulin variable domain in its extracellular domain, the PD-1 intracellular domain contains two tyrosine sites, the common membrane-proximal tyrosine is located in ITIM (immunoreceptor tyrosine inhibitory receptor), and the presence of ITIM receptor on the PD-1 protein indicates that this protein molecule attenuates antigen receptor signaling by recruiting intracellular tyrosine phosphorylase. The sequences of human and murine PD-1 proteins are 60% homologous, and 4 potential N-glycosylation sites and amino acids identified in the variable region of the immunoglobulin are conserved regions. Experiments have shown that PD-1 modulates central and peripheral immune responses in the body by interacting with its ligands, and in particular in the presence of PD-L1, inhibits the proliferation of wild type T cells, but not PD-1 deficient T cells. Moreover, PD-1 deficient mice also exhibit autoimmune responses, and the PD-1 deficient mouse strain C57BL/6 causes, for example, chronic progressive lupus erythematosus, glomerulonephritis and arthritis; balb/c mice with a PD-1 deficiency can cause severe cardiomyopathy due to the presence of cardiac tissue-specific autoantibodies.

since PD-1 plays an extremely important role in autoimmunity, tumor immunity, and infectious immunity, PD-1 is an ideal target for immunotherapy. Therefore, blocking of PD-1 by antagonists, including monoclonal antibodies, has been investigated for the treatment of cancer and chronic viral infections. However, there is still a need to develop new functional specific antibodies with higher affinity for binding human programmed death-1 (PD-1) protein targets.

Disclosure of Invention

Therefore, the present invention provides a human anti-programmed death-1 (PD-1) antibody and its application to solve the above technical problems.

In order to solve the technical problems, the technical scheme of the invention is as follows:

The invention provides a human anti-programmed death-1 (PD-1) antibody or antigen binding fragment, which comprises a heavy chain variable region, wherein the heavy chain variable region comprises at least one amino acid sequence shown as SEQ ID NO 1-2, SEQ ID NO 3-5, SEQ ID NO 6-8, SEQ ID NO 9-11 and SEQ ID NO 12.

Preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO 1-12.

Preferably, the antibody or antigen-binding fragment further comprises a light chain variable region comprising an amino acid sequence as set forth in at least one of SEQ ID NOS 13-14, 15-17, 18-20, 21-23, and 24.

Preferably, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO 13-24.

Preferably, the antibody or antigen-binding fragment comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO 1-12 and the light chain variable region of the amino acid sequence shown in SEQ ID NO 13-24.

Preferably, the antibody or antigen-binding fragment comprises at least one of:

r1: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 1 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 13;

R2, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 2 and a light chain variable region having the amino acid sequence shown in SEQ ID NO. 14;

R3: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 3 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 15;

r4: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 4 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 16;

r5: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 5 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 17;

r6: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 6 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 18;

r7: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 7 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 19;

R8: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 8 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 20;

R9: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 9 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 21;

R10: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 10 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 22;

r11: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 11 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 23;

R12: the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 12 and the light chain variable region having the amino acid sequence shown in SEQ ID NO. 24.

Alternatively, and preferably, the antibody or antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NOS 25-36.

preferably, the antibody or antigen fragment further comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NOS 37-48.

Preferably, the antibody or antigen-binding fragment comprises at least one of:

h1: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 25 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 37;

H2: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 26 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 38;

h3: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 27 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 39;

H4: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 28 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 40;

h5: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 29 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 41;

H6: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 30 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 42;

H7: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 31 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 43;

H8: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 32 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 44;

H9: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 33 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 45;

h10: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 34 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 46;

H11: a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 35 and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 47;

H12: the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 36 and the light chain variable region having the amino acid sequence shown in SEQ ID NO. 48.

Preferably, the antibody or antigen-binding fragment further comprises a humanized antibody or human sequence framework region.

Preferably, the antibody or antigen binding fragment specifically binds to an extracellular epitope of a PD-1 protein of human or murine origin.

Preferably, the heavy chain constant region of the human antibody IgG1, IgG2, IgG3 or IgG4 is also included.

The invention also provides application of the humanized anti-programmed death-1 (PD-1) antibody or antigen binding fragment in-vitro detection of PD-1 protein.

the invention also provides application of the humanized anti-programmed death-1 (PD-1) antibody or antigen binding fragment in a medicine for enhancing T cell immune response.

The invention also provides an application of the human anti-programmed death-1 (PD-1) antibody or antigen binding fragment in a medicine for treating autoimmune diseases, inflammatory diseases, allergy, graft rejection, cancer or immune system disorder.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the human anti-programmed death-1 (PD-1) antibody or antigen binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises at least one amino acid sequence shown as SEQ ID NO 1-2, SEQ ID NO 3-5, SEQ ID NO 6-8, SEQ ID NO 9-11 and SEQ ID NO 12. The antibody or the antigen binding fragment has bioactivity of inducing interleukin-2 release in a mixed lymphocyte experiment, can be combined with a human-derived programmed death-1 (PD-1) protein target with high affinity, can be widely applied to in vitro detection of PD-1 protein, and can be used for preparing a medicament for enhancing T cell immune response and treating autoimmune diseases, inflammatory diseases, allergic reactions, graft rejection, cancers or immune system disorder medicaments.

Drawings

in order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is the DNA sequence and amino acid sequence of the heavy chain variable region (H-CVR) of recombinant PD-1 antibody Abs-r-923 in example 3 of the present invention;

FIG. 2 shows the DNA sequence and amino acid sequence of the heavy chain variable region (H-CVR) of recombinant PD-1 antibody Abs-r-924 in example 3 of the present invention;

FIG. 3 shows the DNA sequence and amino acid sequence of the heavy chain variable region (H-CVR) of recombinant PD-1 antibody Abs-r-925 in example 3 of the present invention;

FIG. 4 shows the DNA sequence and amino acid sequence of the heavy chain variable region (H-CVR) of recombinant PD-1 antibody Abs-r-926 in example 3 of the present invention;

FIG. 5 shows the DNA sequence and amino acid sequence of the light chain variable region (L-CVR) of the recombinant PD-1 antibody Abs-r-923 in example 3 of the present invention;

FIG. 6 shows the DNA sequence and amino acid sequence of the light chain variable region (L-CVR) of recombinant PD-1 antibody Abs-r-924 in example 3 of the invention;

FIG. 7 shows the DNA sequence and amino acid sequence of the light chain variable region (L-CVR) of recombinant PD-1 antibody Abs-r-925 in example 3 of the present invention;

FIG. 8 shows the DNA sequence and amino acid sequence of the light chain variable region (L-CVR) of recombinant PD-1 antibody Abs-r-926 in example 3 of the present invention;

FIG. 9 shows the DNA sequence and amino acid sequence of the heavy chain variable region (H-CVR) of the humanized PD-1 antibody Abs-H-923 of example 3 of the present invention;

FIG. 10 shows the DNA sequence and amino acid sequence of the heavy chain variable region (H-CVR) of the humanized PD-1 antibody Abs-H-924 in example 3 of the present invention;

FIG. 11 shows the DNA sequence and amino acid sequence of heavy chain variable region (H-CVR) of humanized PD-1 antibody Abs-H-925 in example 3 of the present invention;

FIG. 12 shows the DNA sequence and amino acid sequence of the heavy chain variable region (H-CVR) of the humanized PD-1 antibody Abs-H-926 in example 3 of the present invention;

FIG. 13 shows the DNA sequence and amino acid sequence of the light chain variable region (L-CVR) of the humanized PD-1 antibody Abs-h-923 of example 3 of the present invention;

FIG. 14 shows the DNA sequence and amino acid sequence of the light chain variable region (L-CVR) of the humanized PD-1 antibody Abs-h-924 in example 3 of the present invention;

FIG. 15 shows the DNA sequence and amino acid sequence of the light chain variable region (L-CVR) of the humanized PD-1 antibody Abs-h-925 in example 3 of the present invention;

FIG. 16 shows the DNA sequence and amino acid sequence of the light chain variable region (L-CVR) of the humanized PD-1 antibody Abs-h-926 in example 3 of the present invention;

FIG. 17 shows the results of competitive comparison of the four humanized PD-1 antibodies of example 6 of the present invention with Nivolumab;

FIG. 18 is a graph showing the results of comparing the IL-2 release-inducing effects of the humanized PD-1 antibody in example 7 of the present invention.

Detailed Description

Example 1

this example provides a human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment, or, as a variant, a rabbit antibody or functional antigen-binding fragment thereof, in particular a recombinant antibody, also a humanized antibody.

the antibody or antigen binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises at least one amino acid sequence shown as SEQ ID NO 1-2, SEQ ID NO 3-5, SEQ ID NO 6-8, SEQ ID NO 9-11 and SEQ ID NO 12.

The antibody or antigen binding fragment further comprises a light chain variable region comprising at least one amino acid sequence shown as SEQ ID NO 13-14, SEQ ID NO 15-17, SEQ ID NO 18-20, SEQ ID NO 21-23, and SEQ ID NO 24.

Preferably, in this embodiment, the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3 or variants thereof having the amino acid sequences set forth in SEQ ID Nos. 1-3; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3 having the amino acid sequences shown in SEQ ID Nos. 13-15 or variants thereof.

alternatively, in this embodiment, the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3 or variants thereof having the amino acid sequences set forth in SEQ ID Nos. 4-6; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3 having the amino acid sequences shown in SEQ ID Nos. 16-18 or variants thereof.

Alternatively, in this embodiment, the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3 having the amino acid sequences shown in SEQ ID Nos. 7-9, or variants thereof; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3 having the amino acid sequences shown in SEQ ID Nos. 19-21 or variants thereof.

alternatively, in this embodiment, the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3 or variants thereof having the amino acid sequences set forth in SEQ ID Nos. 10-12; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3 having the amino acid sequences shown in SEQ ID Nos. 22-24 or variants thereof.

As described previously, the PD-antibody or antigen-binding fragment thereof can be a rabbit antibody or fragment thereof, wherein the rabbit antibody is selected from abs-r-923, abs-r-924, abs-r-925, and abs-r-926, and the antibody heavy chain variable region further comprises the heavy chain FR region of a rabbit IgG or a variant thereof.

In a preferred embodiment of the invention, the rabbit antibody or fragment thereof further comprises a heavy chain constant region of rabbit IgG or a variant thereof.

Further, in this embodiment, the rabbit-derived antibody or fragment thereof further comprises a light chain FR region of a rabbit-derived kappa chain or a variant thereof.

in a preferred embodiment of the invention, the rabbit antibody or fragment thereof further comprises a light chain constant region of a rabbit λ chain or a variant thereof.

Alternatively, in a preferred embodiment, the PD-1 antibody or antigen-binding fragment thereof is a chimeric antibody or fragment thereof. The heavy chain variable region of the chimeric antibody comprises amino acid sequences shown as SEQ ID NOs:50, 54, 58 and 62.

in the chimeric antibody or a fragment thereof, the light chain variable region of the chimeric antibody comprises the amino acid sequences shown as SEQ ID NOs:52, 56, 60 and 64.

Alternatively, the PD-1 antibody described in this example is a humanized antibody or a functional antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising H-CDR1, H-CDR2 and H-CDR3 or variants thereof of the amino acid sequences shown in SEQ ID Nos. 25-36 and a light chain variable region comprising L-CDR1, L-CDR2 and L-CDR3 or variants thereof of the amino acid sequences shown in SEQ ID Nos. 37-39.

As a switchable embodiment, the heavy chain variable region in the humanized antibody or functional fragment thereof comprises H-CDR1, H-CDR2 and H-CDR3 of the amino acid sequence shown in SEQ ID NOS: 28-30 or variants thereof and the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3 of the amino acid sequence shown in SEQ ID NOS: 40-42 or variants thereof.

alternatively, the heavy chain variable region in the humanized antibody or functional fragment thereof comprises H-CDR1, H-CDR2 and H-CDR3 of the amino acid sequence shown in SEQ ID NO. 31-33 or variants thereof and the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3 of the amino acid sequence shown in SEQ ID NO. 43-45 or variants thereof.

alternatively, the heavy chain variable region in the humanized antibody or functional fragment thereof comprises H-CDR1, H-CDR2 and H-CDR3 of the amino acid sequence shown in SEQ ID NO. 34-36 or variants thereof and the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3 of the amino acid sequence shown in SEQ ID NO. 46-48 or variants thereof.

wherein the humanized antibody is selected from hu923, hu924, hu925 and hu 926.

Preferably, the humanized antibody or fragment thereof, the humanized antibody heavy chain variable region further comprises a heavy chain FR region of human IgG1, IgG2, IgG3, IgG4 or variants thereof.

Preferably, the humanized antibody further comprises a heavy chain constant region.

Preferably, the humanized antibody or fragment thereof, humanized antibody light chain variable region further comprises human kappa, lambda chains or variants thereof.

Preferably, the humanized antibody further comprises light chain FR region sequences.

In a preferred embodiment of the invention, the humanized antibody or fragment thereof is a chimeric antibody or fragment thereof further comprising a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a human IgG1 or IgG4 heavy chain constant region. The chimeric antibody or fragment thereof further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.

In a preferred embodiment of the present invention, among the PD-1 humanized antibody or a fragment thereof, a PD-1 humanized antibody or a fragment thereof.

In this example, a PD-1 chimeric antibody or a fragment thereof, in which the heavy chain variable region of the chimeric antibody comprises the amino acid sequences shown in SEQ ID NOs:66, 70, 74 and 78 and the light chain variable region of the chimeric antibody comprises the amino acid sequences shown in SEQ ID NOs: 68, 72, 76 and 80.

In a preferred embodiment of the invention, in the PD-1 antibody or antigen-binding fragment thereof, the antigen-binding fragment is Fab, Fv, scFv, F (ab') 2.

Further, the present example also provides a polynucleotide (DNA) molecule encoding the above-described PD-1 antibody or antigen-binding fragment thereof, or a combination of isolated polynucleotides, comprising a polynucleotide encoding the light chain of the anti-PD-1 antibody or functional fragment thereof of the present invention and a polynucleotide encoding the heavy chain of the anti-PD-1 antibody or functional fragment thereof of the present invention.

the present example also provides a bispecific molecule comprising an anti-PD-1 antibody or fragment thereof of the invention. The antibodies of the invention or antigen binding portions thereof can be derivatized or linked to another functional molecule, such as another antibody or peptide or protein to generate bispecific molecules that bind to at least two different binding sites or target molecules. The antibodies of the invention may in fact be derivatized or linked to one or more other functional molecules to generate multispecific molecules that bind to two or more different binding sites and/or target molecules.

furthermore, the present embodiment also provides an expression vector containing the above DNA molecule.

And further provides a host cell transformed with the expression vector as described above, preferably a hamster ovary cell, particularly a Chinese hamster ovary Cell (CHO).

the anti-PD-1 antibody or a functional fragment thereof of the invention blocks the interaction of PD-1 and PD-L1 and/or the interaction of PD-1 and PD-L2.

The human anti-programmed death-1 (PD-1) antibody or antigen binding fragment described in this example can be used for in vitro detection of PD-1 protein and can also be used in drugs that enhance T cell immune responses, including enhancement of cytokine production by T cells, preferably by cytokines such as IL-2. And also has the function of competitively binding to PD-1 protein, in a specific embodiment, the characteristic of competitively binding to human PD-1 protein is preferably Nivolumab (Nivolumab). The human anti-programmed death-1 (PD-1) can also be used in drugs for treating autoimmune diseases, inflammatory diseases, allergies, graft rejection, cancer or immune system disorders.

In this example, the H-CDR and complete heavy chain variable sequences of the PD-1 antibody Abs-r-923 are shown in Table 1.

TABLE 1

The L-CDR and complete light chain variable sequences of the PD-1 antibody Abs-r-923 are shown in Table 2.

TABLE 2

the H-CDR and complete heavy chain variable sequences of the PD-1 antibody Abs-H-923 are also shown in Table 3.

TABLE 3

The L-CDR and complete light chain variable sequences of the PD-1 antibody Abs-h-923 are also shown in Table 4.

TABLE 4

The H-CDR and complete heavy chain variable sequences of the PD-1 antibody Abs-r-924 are shown in Table 5.

TABLE 5

The L-CDR and complete light chain variable sequences of the PD-1 antibody Abs-r-924 are shown in Table 6.

TABLE 6

The H-CDR and complete heavy chain variable sequences of the PD-1 antibody Abs-H-924 are also shown in Table 7.

TABLE 7

The L-CDR and complete light chain variable sequences of the PD-1 antibody Abs-h-924 are also shown in Table 8.

TABLE 8

the H-CDR and complete heavy chain variable sequences of the PD-1 antibody Abs-r-925 are shown in Table 9.

TABLE 9

the L-CDR and complete light chain variable sequences of the PD-1 antibody Abs-r-925 are shown in Table 10.

Watch 10

The H-CDR and complete heavy chain variable sequences of the PD-1 antibody Abs-H-925 are also shown in Table 11.

TABLE 11

The L-CDR and complete light chain variable sequences of the PD-1 antibody Abs-h-925 are also shown in Table 12.

TABLE 12

The H-CDR and complete heavy chain variable sequences of the PD-1 antibody Abs-r-926 are shown in Table 13.

watch 13

the L-CDR and complete light chain variable sequences of the PD-1 antibody Abs-r-926 are shown in Table 14.

TABLE 14

the H-CDR and complete heavy chain variable sequences of the PD-1 antibody Abs-H-926 are also shown in Table 15.

watch 15

the L-CDR and complete light chain variable sequences of the PD-1 antibody Abs-h-926 are also shown in Table 16.

TABLE 16

Example 2

preparation of Rabbit anti-PD-1 antibody

a. Animal immunization

4 female rabbits 4 months old were immunized and each rabbit was injected subcutaneously with 50. mu.g of recombinant human PD-1(Sigma-Aldrich, Cat #263810) mixed with complete Freund's adjuvant. Two to three weeks later, rabbits were inoculated with 25. mu.g of soluble PD-1 and incomplete Freund's adjuvant (Sigma-Aldrich, Cat #263910), auricular venous blood of the rabbits was collected to examine immunoreaction, serum was isolated, followed by dilution with PBS1/1000 and ELISA examination was performed to analyze whether or not the titer was enhanced, after which rabbits were selected to obtain a sufficient amount of antibody, and the selected rabbits were subjected to a cell fusion process.

b. Preparation of splenocytes and cell fusions

The rabbit spleen was extracted and a single cell suspension of pooled spleens was prepared. Spleen cells and myeloma cells were washed several times with DMEM and fused 2 times in the presence of 1ml of 50% (w/v) PEG 3550 (ratio of spleen cells to SP2/0 was 2.5:1 and 2.4: 1). The resulting hybridomas were resuspended in CGM (HAT medium) containing 20% FCS and aminopterin. Cell suspensions (140 Cl/well) of each fusion were plated in CGM containing 20% FCS to 8 96-well tissue culture flat-bottom plates (Corning-Costar) containing 140 Cl/well peritoneal effusion cells as feeder cells. Plates were incubated for 10 days. During this period, cells were fed 2 times with HAT medium. Aliquots of spleen cell preparations (approximately 8X 10)⁶Individual splenocytes) were cultured in wells of 24-well plates for 10 days and cell culture supernatant was used as a positive control in ELISA.

c. screening assays

The cell culture supernatants were screened for IgG using ELISA assay. 96-well flat-bottomed polystyrene microtiter plates (Greiner, Cat #655061) were coated with 50. mu.l/well of PD-1Fc antigen at pH 9.6 (5. mu.g/ml) in 0.5M carbonate/bicarbonate buffer. After overnight incubation at 4 ℃ in a humidity chamber, plates were washed with Tris-buffered saline (TBS,50mM Tris, pH 7.8,500mM sodium chloride) (wash buffer) containing 0.01% Triton X-100 and then incubated with 200. mu.l/well 2% FCS (blocking buffer) in TBS for 1 hour at Room Temperature (RT) on a shaker. The wells were washed with wash buffer and 100. mu.l of cell culture supernatant was added to the appropriate wells. Cell culture supernatants from SP2/0 myeloma cells were used as negative controls. Cell culture supernatants from spleen cell cultures were used as positive controls. Plates were incubated on a shaker at RT for 1 hour and then washed several times. To detect bound antibodies, plates were incubated for 1 hour at RT on a shaker with 50. mu.l/well of alkaline phosphatase conjugated goat anti-rabbit IgG in blocking buffer, washed several times, and 150. mu.l/well of substrate buffer (5% diethanolamine +0.5mM MgCl2, 2mM 4-nitrophenyl phosphate in pH 9.8) was added. The absorbance values (OD) were read at 405nm in a Dynex Opsys MR microplate reader.

d. monoclonal selection

Cells from positive IgG-producing cultures were transferred to wells of 48-well plates and cultured for several days (depending on the growth characteristics of the cells). ELISA (on PD-1Fc and without pre-coated antigen) was performed to select specific binders. Cells from ELISA-positive wells were frozen in freezing medium (90% FCS, 10% DMSO). Aliquots of the cells were further cultured to produce cell culture supernatants for further characterization. Once positive wells are identified, hybridoma cells are cloned to reduce the risk of overgrowth of non-producer cells (first cloning). To ensure that the antibody is truly monoclonal, the hybridoma is again cloned (second clone). Limiting dilution method was used for both cloning procedures. IgG-producing cells were distributed into a 96-well plate containing feeder cells at a density of 1-3 cells per well. After 8-10 days (depending on growth characteristics), all plates were visually observed under a microscope to detect monoclonal growth. Using the screening assays described above, culture supernatants from these wells were screened for specific immunoglobulin content. Appropriate clones were selected for growth characteristics and ELISA signal, transferred to wells of a 24-well plate, and cultured for several days for screening assays. This process was repeated 2 to 3 times. The appropriate subclones were selected for secondary cloning or for culture (for cryopreservation), respectively. The 4 screened anti-PD-1 antibodies with strong specificity and binding force are respectively named as Abs-r-923, Abs-r-924, Abs-r-925 and Abs-r-926, and are subjected to specificity detection, and are not bound with other non-specific antibodies and do not cross-react with the mouse PD-1.

example 3

Humanization of PD-1 antibodies

a. Humanized heavy chain variable region sequence

Four PD-1 antibodies were humanized by screening human open reading sequences with low immunogenicity by comparing the known human immunoglobulin heavy chain variable region sequences, including Abs-r-923, Abs-r-924, Abs-r-925, and Abs-r-926. The DNA and amino acid sequences of the four recombined antibodies are shown in the figures 1-4; SEQ ID NOS:49, 50, 53, 54, 57, 58, 61 and 62 can also be queried.

The DNA and amino acid sequences of the heavy chain variable regions of humanized Abs-h-923, Abs-h 924, Abs-h 925, Abs-h 926 can be queried as SEQ ID NOS 65, 66, 69, 70, 73, 74, 77 and 78.

Four PD-1 antibodies were humanized by screening human open reading sequences with low immunogenicity by comparing the known human immunoglobulin light chain variable region sequences, including Abs-r-923, Abs-r-924, Abs-r-925, and Abs-r-926. The DNA and amino acid sequences of the four recombinant antibodies are shown in FIGS. 5-8; SEQ ID NOS:51, 52, 55, 56, 59, 60, 63 and 64 can also be queried.

The DNA and amino acid sequences of the heavy chain variable region of humanized Abs-h-923, Abs-h 924, Abs-h 925 and Abs-h 926 are shown in FIGS. 9-12.

The DNA and amino acid sequences of the light chain variable regions of humanized Abs-h-923, Abs-h 924, Abs-h 925 and Abs-h 926 are shown in FIGS. 13-16, and SEQ ID NOS:67, 68, 71, 72, 75, 76, 79 and 80 can also be referred to.

example 4

construction and expression of humanized PD-1 antibodies

Human IgG4 heavy chain constant region fragment IgG4Fc sequence and k light chain constant region sequence were synthesized by IDT corporation (Integrated DNA Technologies, Coralville, Lowa), the synthesized fragments were ligated to pcDNA3.1 vector, plasmid vectors were transformed into pBA-H4 and pBA-Ck, pBA-H4 (containing human IgG4 heavy chain constant region fragment IgG4Fc), pBA-Ck (containing human k light chain constant region), plasmid vectors were constructed by Bioabs corporation, plasmids were expressed in CHO cells, and then DNA sequences encoding heavy chain and light chain variable regions were further modified, the DNA sequences encoding the heavy chain variable region of the humanized anti-PD-1 antibody described in the present invention were set forth in SEQ ID Nos:65(Abs-H-923), 69(Abs-H-924),73(Abs-H-925) and 77(Abs-H-926), the DNA sequence encoding the heavy chain variable region of the humanized anti-PD-1 antibody of the present invention was set forth in SEQ ID Nos:67(Abs-H-923), 71(Abs-h-924),75(Abs-h-925) and 79 (Abs-h-926).

Example 5

Affinity of humanized antibodies

The SPR BacoreTM technology is used for detecting the affinity of PD-1 antibodies Ab-h-923, Ab-h-924, Ab-h-925 and Ab-h-926, and is particularly suitable for detecting antigen-antibody interaction. The anti-PD-1 antibody was diluted in the detection buffer at a concentration of two times, from a concentration range of 0.14 to 7.68nM, and its association kinetic rate constant (Ka), dissociation kinetic rate constant (Kd), and dissociation equilibrium constant (KD) were measured. The results of the affinity to Abs-h-923, Abs-h-924, Abs-h-925, Abs-h-926 are shown in Table 17.

TABLE 17

example 6

Comparison of the competitive Properties of humanized PD-1 antibody with Nivolumab

The competition of Abs-h-923, Abs-h-924, Abs-h-925, Abs-h-926 and Nivolumab was analyzed by ELISA assay. The specific method comprises the following steps: a series of different concentrations of Abs-h-923, Abs-h-924, Abs-h-925, Abs-h-926PD-1 antibody, 50ng/ml Nivolumab or a control antibody (negative control) were preincubated at 37 ℃ for 1 hour, the mixture was added to a 96-well plate coated with 100ng/ml human PD-1 and incubated for an additional 1 hour, washed, HRP was added, incubation, TMB was added, after which the reaction was stopped and absorbance values (OD) were read at 450 nm.

The humanized PD-1 antibody has the property of binding competitively to human PD-1 protein with Nivolumab. However, Abs-h-923 was less competitive with Abs-h-926 being the most competitive, and the control antibody failed to compete with Nivolumab for PD-1, as shown in FIG. 17.

Example 7

humanized PD-1 antibodies induce IL-2 release

Four antibodies: abs-h-923, Abs-h-924, Abs-h-925 and Abs-h-926 were tested for their enhancement of IL-2 release by the Mixed Lymph Reaction (MLR) method. This experiment was performed using Nivolumab as a control. DC cells were used as stimulator cells, purified CD4+ T lymphocytes were used as effector cells, and supernatants were collected 2.5 days later for assay.

As shown in FIG. 18, after treatment with PD-1 antibodies Abs-h-923, Abs-h 924, Abs-h 925 and Abs-h 926, the release of IL-2 factor can be significantly enhanced, wherein the induction of Abs-h 923 is the weakest and the induction of Abs-h 926 is similar to that of Nivolumab.

it should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. A human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment, comprising a heavy chain variable region comprising an amino acid sequence set forth in at least one of SEQ ID NOS 1-2, 3-5, 6-8, 9-11, and 12.

2. The human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment of claim 1, wherein said heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NOs 1-12.

3. The human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment of claim 1 or 2, further comprising a light chain variable region comprising an amino acid sequence set forth in at least one of SEQ ID NOS 13-14, 15-17, 18-20, 21-23, and 24.

4. The human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment of claim 3, wherein said light chain variable region comprises an amino acid sequence as set forth in SEQ ID NOS: 13-24.

5. The human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment of claim 4, wherein said antibody or antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NOS: 1-12 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NOS: 13-24.

6. the human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment of claim 5, wherein said antibody or antigen-binding fragment includes at least one of:

7. the human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment of claim 1, wherein said antibody or antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NOS: 25-36.

8. the human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment of claim 7, further comprising a light chain variable region having an amino acid sequence as set forth in SEQ ID NOS: 37-48.

9. the human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment of claim 8, wherein said antibody or antigen-binding fragment comprises at least one of:

10. The human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment of any one of claims 1-9, further comprising a humanized antibody or human sequence framework region.

11. The human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment of claim 10, wherein said antibody or antigen-binding fragment specifically binds to an extracellular epitope of a human or murine PD-1 protein.

12. The human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment of claim 11, further comprising a heavy chain constant region of human antibody IgG1, IgG2, IgG3, or IgG 4.

13. use of the human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment of any one of claims 1-12 for the in vitro detection of PD-1 protein.

14. Use of a human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment according to any one of claims 1 to 12 in a medicament for enhancing a T cell immune response.

15. Use of a human anti-programmed death-1 (PD-1) antibody or antigen-binding fragment according to any one of claims 1 to 12 in a medicament for treating an autoimmune disease, an inflammatory disease, an allergy, a transplant rejection, a cancer, or an immune system disorder.