CN107840887A - A new PD‑1 monoclonal antibody - Google Patents

Abstract

The present invention provides monoclonal antibodies to PD-1, particularly human monoclonal antibodies to PD-1, which specifically bind to PD-1 with high affinity and comprise a heavy chain and a light chain. The invention also provides nucleic acid sequences encoding the antibodies of the invention, cloning or expression vectors, host cells, methods for expressing or isolating the antibodies, immunoconjugates comprising the antibodies of the invention and therapeutic compositions. The invention further provides the use of the PD-1 antibodies to treat various cancers.

Description

A new PD-1 monoclonal antibody

technical field

The present invention mainly relates to PD-1 monoclonal antibody and its composition, and the immunotherapy of human diseases using anti-PD-1 antibody.

Background technique

Mounting evidence of preclinical and clinical outcomes suggests that targeting immune checkpoints is emerging as the most promising approach for treating cancer patients. Programmed cell death molecule 1 (PD-1), an inhibitory member of the immunoglobulin superfamily with homology to CD28, is expressed in activated B cells, T cells, and myeloid cells (Agata et al, supra; Okazaki et al(2002) Curr.Opin.Immunol.14:391779-82; Bennett et al.(2003) JImmunol 170:711-8) and play an important role in regulating activating and inhibitory signals in the immune system (Okazaki, Taku et al. 2007 International Immunology 19:813-824). In fact, PD-1 was discovered in a differential expression screen of apoptotic cells (Ishida et al (1992) EMBO J 11:3887-95).

The structure of PD-1 is a monomer type I transmembrane protein, belonging to the Ig gene superfamily (Agata et al. (1996) bitImmunol 8:765-72), which consists of an immunoglobulin variable region-like extracellular domain and Consists of a cytoplasmic domain containing an immunoreceptor tyrosine-inhibiting motif (ITIM) and an immunoreceptor tyrosine-switching motif (ITSM). Despite its structural similarity to CTLA-4, PD-1 lacks the MYPPPY motif that binds B7-1 and B7-2. PD-1 has two known ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), which are members of the B7 family expressed on the cell surface (Freeman et al (2000) JExp Med 192:1027-34; Latchman et al (2001) Nat Immunol 2:261-8; Carter et al (2002) EurJ Immunol 32:634-43). PD-L1 and PD-L2, which are homologous to B7, bind to PD-1 but not to other CD28 family members.

PD-1, as one of the immune checkpoint proteins, is an inhibitory member of the immunoglobulin superfamily with homology to CD28, expressed in activated B cells, T cells and myeloid cells (Agata et al, supra; Okazaki et al.(2002) Curr Opin Immunol 14:391779-82; Bennett et al.(2003) JImmunol 170:711-8), and play an important role in limiting the activation of T cells, which provides an opportunity for tumor cells to escape immune surveillance important immune mechanism. PD-1 induces T cell anergy, or a state of anergy, resulting in a temporary inability to produce optimal levels of effector cytokines within the cells. PD-1 can also induce apoptosis of T cells through its ability to suppress survival signals. PD-1-deficient animals develop various autoimmune phenotypes, including autoimmune cardiomyopathy and arthritic and nephritic lupus-like syndromes (Nishimura et al. (1999) Immunity 11:141-51; Nishimura et al. (2001) Science 291:319-22). Furthermore, PD-1 has been found to play an important role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type I diabetes, and rheumatoid arthritis (Salama et al. (2003) JExp Med 198:71-78: Prokunina and Alarcon-Riquelme (2004) Hum MoI Genet 13:R143; Nielsen et al. (2004) Lupus 11:510). In a mouse B-cell tumor line, the ITSM of PD-1 was shown to be essential for blocking BCR-mediated phosphorylation of Ca ²⁺ -flux and tyrosine downstream effector molecules (Okazaki et al. (2001 ) PNAS 98:13866-71).

Interactions between PD-1 expressed on activated T cells and PD-L1 expressed on tumor cells negatively regulate immune responses and reduce antitumor immunity. PD-L1 is expressed in various human cancers (Dong et al (2002) Nat. Med 8:787-9). In esophageal, pancreatic, and other types of cancer, tumor expression of PD-L1 was associated with reduced survival, highlighting this pathway as a promising new target for cancer immunotherapy. Several groups have shown that PD-1-PD-L interaction exacerbates disease, leading to a reduction in tumor-infiltrating lymphocytes, a reduction in T cell receptor-mediated proliferation, and immune evasion of cancerous cells (Dong et al.( 2003) J. MoI. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immunosuppression can be reversed by inhibiting the local interaction of PD-L1 and PD-1, the effects of which are cumulative when the interaction of PD-1 and PD-L2 is blocked.

Pharmaceutical companies have developed a variety of drugs targeting the PD-1/PD-L1 pathway, such as Bristol-Myers Squibb (BMS), Merck, Roche, and GlaxoSmithKline (GSK), among others. Data from clinical trials showed early evidence of durable clinical activity and a favorable safety profile in patients with various tumor types. At present, three antibody drugs targeting the PD-1/PD-L1 pathway have been approved internationally, namely Nivolumab from BMS, Pembrolizumab from Merck and Atezolizumab from Roche. Nivolumab, an anti-PD-1 drug developed by BMS, is being put into the center stage of the next-generation field. In six late-stage studies now, treatment led to tumor shrinkage in three of five cancer groups studied, including 18 percent of 72 lung cancer patients and nearly one-third of 98 melanoma patients One and 27% of the 33 patients with kidney cancer. Pembrolizumab, developed by Merck, is a fully human monoclonal IgG4 antibody that acts on PD-1, and its impressive IB data against skin cancer has reached the FDA's new breakthrough indicator. The results of the phase IB study showed 51% antitumor response and 9% complete response in 85 cancer patients. Meanwhile, the overall response rates of pembrolizumab in patients with head and neck cancer, gastric cancer, and urothelial cancer were 21.4%, 22.2%, and 27.6%, respectively. Roche's Atezolizumab, a fully human monoclonal IgG1 antibody targeting PD-L1, was shown to shrink tumors in 29 (21%) of 140 patients with advanced cancers of various sizes Advanced urothelial carcinoma patients with high expression of PD-L1 on the cell surface showed a higher tumor suppression effect (27%).

Not all existing treatments are satisfactory. Most PD-1 antibody drugs do not bind to the mouse PD-1 protein, which limits the application of antibody drugs in preclinical animal experiments, and because most of their antibody sequences are derived from immunity to mice, serious Immunogenic reactions reduce the therapeutic effect of antibody drugs in humans. A humanized antibody cross-reactive with mouse PD-1 overcomes these shortcomings and exhibits better tolerance and higher efficacy in vivo. Therefore, novel anti-PD-1 antibodies are still needed.

Contents of the invention

The invention provides isolated antibodies, particularly monoclonal antibodies or human monoclonal antibodies.

In one aspect, the present invention provides an antibody or antigen-binding fragment thereof, which binds to an epitope of PD-1, the epitope comprising: 128, 129, 130, 131 and 132 on SEQ ID NO: 24 point amino acid and at least one amino acid in positions 35, 64, 82, and 83.

The present invention also provides an antibody or an antigen-binding fragment thereof, which binds to an epitope of human PD-1 and mouse PD-1, wherein the epitope includes 128th, 129th, and 130th on SEQ ID NO: 24 , 131 and 132 amino acids.

The antibody or antigen-binding fragment thereof as described above, wherein the mouse PD-1 is mouse or rat PD-1.

In some embodiments, the antibody in the above-mentioned antibody or antigen-binding fragment thereof

a) bind to human _PD -1 with a KD of 2.15E-10M or less; and

b) Binding to mouse PD-1, K _D is below 1.67E-08M.

In some embodiments, the aforementioned antibodies have at least one of the following properties:

a) Binding to human PD-1 with a K _D of 4.32E-10M to 2.15E-10M, and binding to mouse PD-1 with a K _D of 5.39E-08M to 1.67E-08M;

b) substantially not binding to human CD28, CTLA-4;

c) increasing the proliferation of T cells;

d) increase the production of interferon-gamma; or

e) Increased secretion of interleukin-2.

The present invention provides an antibody or antigen-binding fragment thereof comprising an amino acid sequence that is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9 The sequences in the group have at least 70%, 80%, 90% or 95% homology,

Wherein the antibody specifically binds to PD-1.

The present invention provides an antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9 in the sequence,

Wherein the antibody specifically binds to PD-1.

The present invention provides an antibody, or an antigen-binding fragment thereof, comprising:

a) a heavy chain variable region whose amino acid sequence is at least 70%, 80%, 90% or 95% homologous to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; as well as

b) a light chain variable region having an amino acid sequence at least 70%, 80%, 90% or 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8 and 9 % homology,

Wherein the antibody specifically binds to PD-1.

The present invention provides an antibody or an antigen-binding fragment thereof, comprising:

a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; and

b) a light chain variable region whose amino acid sequence is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8 and 9,

Wherein the antibody specifically binds to PD-1.

In some embodiments, the antibody comprises:

a) heavy chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 1; and

b) light chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 3,

Wherein the antibody specifically binds to PD-1.

Or in some specific embodiments, the antibody comprises:

a) heavy chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 2; and

b) light chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 3,

Wherein the antibody specifically binds to PD-1.

Or in some specific embodiments, the antibody comprises:

a) heavy chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 2; and

b) a light chain variable region whose amino acid sequence is selected from the sequence shown in SEQ ID NO: 4,

Wherein the antibody specifically binds to PD-1.

Or in some specific embodiments, the antibody comprises:

a) heavy chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 2; and

b) a light chain variable region whose amino acid sequence is selected from the sequence shown in SEQ ID NO: 5,

Wherein the antibody specifically binds to PD-1.

Or in some specific embodiments, the antibody comprises:

a) heavy chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 1; and

b) a light chain variable region whose amino acid sequence is selected from the sequence shown in SEQ ID NO: 6,

Wherein the antibody specifically binds to PD-1.

Or in some specific embodiments, the antibody comprises:

a) heavy chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 1; and

b) a light chain variable region whose amino acid sequence is selected from the sequence shown in SEQ ID NO: 5,

Wherein the antibody specifically binds to PD-1.

Or in some specific embodiments, the antibody comprises:

a) heavy chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 2; and

b) a light chain variable region whose amino acid sequence is selected from the sequence shown in SEQ ID NO: 6,

Wherein the antibody specifically binds to PD-1.

Or in some specific embodiments, the antibody comprises:

a) heavy chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 2; and

b) a light chain variable region whose amino acid sequence is selected from the sequence shown in SEQ ID NO: 7,

Wherein the antibody specifically binds to PD-1.

Or in some specific embodiments, the antibody comprises:

a) heavy chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 1; and

b) a light chain variable region whose amino acid sequence is selected from the sequence shown in SEQ ID NO: 8,

Wherein the antibody specifically binds to PD-1.

Or in some specific embodiments, the antibody comprises:

a) heavy chain variable region, the amino acid sequence of which is selected from the sequence shown in SEQ ID NO: 2; and

b) a light chain variable region whose amino acid sequence is selected from the sequence shown in SEQ ID NO: 9,

Wherein the antibody specifically binds to PD-1.

See Table 1 and sequence listing information for details of the sequence:

Table 1 The specific sequence of the heavy chain and light chain of the antibody

In another aspect, the present invention provides an antibody or an antigen-binding fragment thereof comprising a complementarity determining region (CDR) whose amino acid sequence is selected from the group consisting of SEQ ID NOs: 10-23,

Wherein the antibody specifically binds to PD-1.

In another aspect, the present invention provides an antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences; and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences,

Wherein the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13 and conservative modifications thereof,

Wherein the antibody specifically binds to PD-1.

The light chain variable region CDR3 sequence of the above antibody preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22 and 23 and conservative modifications thereof.

The heavy chain variable region CDR2 sequence of the above antibody preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and conservative modifications thereof.

The light chain variable region CDR2 sequence of the above antibody preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19 and conservative modifications thereof.

The heavy chain variable region CDR1 sequence of the above antibody preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and conservative modifications thereof.

The light chain variable region CDR1 sequence of the above antibody preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17 and 18 and conservative modifications thereof.

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences; and

A light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 10,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 11,

c) a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13,

d) light chain variable region CDR1, the sequence comprising the amino acid sequence shown in the group consisting of SEQ ID NO: 14-18,

e) light chain variable region CDR2, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

f) light chain variable region CDR3, the sequence comprising the amino acid sequence shown in the group consisting of SEQ ID NO: 20-23,

Wherein the antibody specifically binds to PD-1.

In some embodiments, the antibody comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 10,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 11,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 12,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 14,

e) light chain variable region CDR2, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 20,

Wherein the antibody specifically binds to PD-1.

In some embodiments, the antibody comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 10,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 11,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 13,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 14,

e) light chain variable region CDR2, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 21,

Wherein the antibody specifically binds to PD-1.

In some embodiments, the antibody comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 10,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 11,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 13,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 15,

e) light chain variable region CDR2, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 21,

Wherein the antibody specifically binds to PD-1.

In some embodiments, the antibody comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 10,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 11,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 13,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 16,

e) light chain variable region CDR2, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 21,

Wherein the antibody specifically binds to PD-1.

In some embodiments, the antibody comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 10,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 11,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 12,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 17,

e) light chain variable region CDR2, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 21,

Wherein the antibody specifically binds to PD-1.

In some embodiments, the antibody comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 10,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 11,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 12,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 16,

e) light chain variable region CDR2, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 21,

Wherein the antibody specifically binds to PD-1.

In some embodiments, the antibody comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 10,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 11,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 13,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 17,

e) light chain variable region CDR2, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 21,

Wherein the antibody specifically binds to PD-1.

In some embodiments, the antibody comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 10,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 11,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 13,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 17,

e) light chain variable region CDR2, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 22,

Wherein the antibody specifically binds to PD-1.

In some embodiments, the antibody comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 10,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 11,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 12,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 18,

e) light chain variable region CDR2, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 23,

Wherein the antibody specifically binds to PD-1.

In some embodiments, the antibody comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 10,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 11,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 12,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 18,

e) light chain variable region CDR2, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 20,

Wherein the antibody specifically binds to PD-1.

See Table 2 and sequence listing information for specific CDR sequences:

Table 2 The specific sequence of the heavy chain and light chain of the antibody

In some embodiments, the antibody is a chimeric antibody or a humanized antibody or a human antibody.

In some embodiments, wherein said antibody exhibits at least one of the following properties:

a) The KD for binding to human _PD -1 is below 2.15E-10M, and the KD for binding to mouse _PD -1 is below 1.67E-08M;

b) substantially not binding to human CD28, CTLA-4;

c) increase T cell proliferation;

d) increase the production of interferon-gamma; or

e) Increased secretion of interleukin-2.

In yet another aspect, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in the present invention.

The present invention provides a cloning or expression vector comprising the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention.

The present invention provides a host cell comprising one or more of the above-mentioned cloning or expression vectors.

In another aspect, the invention provides a process for producing any of the antibodies of the invention, comprising culturing the host cell described in the invention, and isolating the antibody.

The above antibody is prepared by immunizing SD rats with the extracellular domain of human PD-1 and the extracellular domain of mouse PD-1.

The present invention provides a transgenic rat comprising human immunoglobulin heavy chain and light chain transgenes, wherein the rat expresses any antibody described in the present invention.

The present invention provides a hybridoma obtained from the above rat, characterized in that the hybridoma produces the antibody.

In another aspect, the present invention also provides a pharmaceutical composition, which comprises any antibody or antigen-binding fragment thereof described in the present invention, and more than one pharmaceutically acceptable excipient, diluent or carrier.

The invention also provides an immunoconjugate comprising any antibody or antigen-binding fragment thereof of the invention linked to a therapeutic agent.

The present invention also provides a pharmaceutical composition, which comprises the above immunoconjugate and a pharmaceutically acceptable excipient, diluent or carrier.

The present invention also provides a method for preparing an anti-PD-1 antibody or an antigen-binding fragment thereof, comprising:

(a) provide:

(i) comprising a heavy chain variable region antibody sequence comprising a CDR1 sequence selected from SEQ ID NO: 10, a CDR2 sequence selected from SEQ ID NO: 11 and a CDR3 selected from SEQ ID NO: 12 or SEQ ID NO: 13 sequence; and/or

(ii) comprising a light chain variable region antibody sequence comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17 and 18, a CDR2 sequence selected from SEQ ID: 19 and a CDR2 sequence selected from CDR3 sequences in the group consisting of SEQ ID NOs: 20, 21, 22 and 23; and

(b) Expression changes the antibody sequence into a protein.

The invention also provides a method of modulating an immune response in a subject, comprising administering to the subject any antibody or antigen-binding fragment thereof described in the invention.

The present invention also provides the use of any antibody as described in the present invention in the preparation of a medicament for treating or preventing immune disorders or cancer.

The present invention also provides a method of inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of any antibody or antigen-binding fragment thereof described in the present invention, so as to inhibit the growth of tumor cells.

In the present invention, the above-mentioned tumor cells are selected from melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular malignant melanoma Cancers in the group consisting of melanoma, uterine cancer, ovarian cancer, and rectal cancer.

In the present invention, the above-mentioned antibody is a chimeric antibody or a humanized antibody.

Beneficial Effects of the Invention

The beneficial effect of the present invention lies in the production of anti-PD-1 humanized antibodies by proprietary hybridoma technology. The antibody reported in this invention has high binding affinity; specifically binds human and mouse PD-1 proteins without family cross-reactivity; effectively regulates immune responses, including enhancing T cell proliferation and increasing cytokines IFN-γ and IL-2 produce.

The new anti-PD-1 antibody is derived from the immunization of rats, and its combination with mouse PD-1 protein overcomes the deficiency that preclinical experiments cannot be used in mouse animal models; and after the antibody sequence is humanized, Its humanization degree is close to 100%, which greatly reduces the adverse reactions of drugs used in human body.

Description of drawings

FIG. 1 is a graph showing the binding of 16 hybridoma antibodies to human PD-1 or mouse PD-1 on the cell surface. Figure 1A shows the binding of 16 hybridoma antibodies to human PD-1 on the cell surface; Figure 1B shows the binding of hybridoma antibodies to mouse PD-1 on the cell surface.

Figure 2 is a graph showing the results of the first round of mutant library screening. Sequence and analytical mutations of high affinity clones were used for the second round of mutations.

Figure 3A shows the binding curve of the humanized antibody to human PD-1 on the cell surface, and the antibody specifically binds to human PD-1 with an EC50 of 2.20-2.78 nM. Figure 3B shows the binding curve of the humanized antibody to mouse PD-1 on the cell surface, and the antibody specifically binds to mouse PD-1 with an EC50 of 11.8-15.1 nM. Figure 3C shows a graph of binding of humanized antibodies to activated cynomolgus monkey PBMCs. The isotype control is human IgG4kappa. The same below.

Figure 4 shows the results of the ELISA of the species cross-reactivity test between the antibody and human, mouse, and cynomolgus monkey PD-1. Dependent form binding. Figure 4A is the binding of humanized PD-1 antibody to human PD-1 protein; Figure 4B is the binding of humanized PD-1 antibody to mouse PD-1 protein; Figure 4C is the binding of humanized PD-1 antibody to human PD-1 protein Cynomolgus monkey PD-1 protein binding

Figure 5 shows the results of cross-reactivity of humanized antibodies with CD28 and CTLA-4 proteins of the same family as PD-1. The results showed that the antibody specifically bound to PD-1, but not to CD28 and CTLA-4 of the same family as PD-1.

Figure 6A shows that humanized antibodies block the binding of human PD-L1 to human PD-1 on the surface of CHO-S cells, and Figure 6B shows that humanized antibodies block the binding of mouse PD-L1 to the surface of 293F cells. Binding of PD-1.

Figure 7 shows that the humanized antibody blocks the binding of human PD-L2 to PD-1 protein, and the blocking effect is dose-dependent.

8A-8B show the epitope test results showing that the humanized PD-1 antibody binds to the same or similar epitope as the control antibody. Figure 8A shows the epitope that competed with control antibody 1 (WBP305BMK1), and Figure 8B shows the epitope that competed with control antibody 2 (Keytruda).

Figure 9 shows the cross-reactivity of anti-PD-1 antibodies with human/mouse PD-1; 2 μg/mL of each antibody was coated overnight in a 96-well plate and incubated with hPD-1/mPD-1-His protein , and then add HRP-anti-His antibody for detection.

Figure 10 shows the hotspot residues mapped in the HPD-1 structure. (A) hPD-L1 binding site, data obtained from literature Zak et al.2015; (B-C) binding epitopes of antibody W3052_r16.88.9 and Keytruda respectively, data from Table 8; the color of the picture is used to help distinguish between epitopes difference between.

Figure 11 shows a comparison between human and murine PD-1. Its apparent structural differences (BC loop and C'D loop (or C" strand of MPD-1)) are marked in orange. (A) Structure of hPD-1 (PDB code 4ZQK). According to its NMR structure (PDB code 2M2D) and remodeling the missing loop (Asp85-Asp92). (B) Structure of mPD-1 (PDB code 3BIK)

Figure 12 shows the results of human allogeneic mixed lymphocyte reaction (allo-MLR), indicating that anti-PD-1 antibody can enhance the function of human CD4 ⁺ T cells. As shown in Figure 12A, all tested anti-PD-1 antibodies increased human IL-2 secretion in a dose-dependent manner. Figure 12B shows that anti-PD-1 antibody increases the secretion of human IFN-γ in a dose-dependent manner. As shown in FIG. 12C , all tested anti-PD-1 antibodies increased the proliferation level of human CD4 ⁺ T cells in a dose-dependent manner.

Figure 13 shows the results of mouse allogeneic mixed lymphocyte reaction, indicating that anti-PD-1 antibody can enhance the function of mouse CD4 ⁺ T cells. Figure 13A shows that all tested anti-PD-1 antibodies increased mouse IL-2 secretion in a dose-dependent manner. Figure 13B shows that anti-PD-1 antibody increased the secretion of mouse IFN-γ in a dose-dependent manner. As shown in FIG. 13C , all tested anti-PD-1 antibodies increased the proliferation of mouse CD4 ⁺ T cells in a dose-dependent manner.

Figure 14 shows the results of human allogeneic mixed lymphocyte reaction, indicating that PD-1 antibody can enhance the function of human CD4 ⁺ T cells. Figure 14A shows that humanized PD-1 antibodies increase IFN-γ production in specific T cell responses. Figure 14B shows that humanized PD-1 antibody increases the proliferation of CD4 ⁺ T cells.

Figure 15 demonstrates that PD-1 antibody can reverse the suppressive function of Treg. Figure 15A shows that PD-1 antibody restored IFN-γ secretion. Figure 15B shows that PD-1 antibody restored the proliferation of effector T cells.

Figure 16 shows the results of the ADCC assay, demonstrating that anti-PD-1 antibodies do not mediate the ADCC activity of activated CD4 ⁺ T cells.

Figure 17 shows the results of the CDC assay, demonstrating that anti-PD-1 antibodies do not mediate the CDC activity of activated CD4 ⁺ T cells.

Figure 18 shows the body weight changes of mice in different groups. Changes in body weight of the CloudmanS91 homograft model tumor-bearing mice after administration of 2E5. Data points represent mean body weight within groups and error bars represent standard error (SEM).

Fig. 19 shows relative body weight change (%). Relative body weight changes were calculated based on animal body weight at the start of dosing. Data points represent the mean percent change in body weight within the group and error bars represent standard error (SEM).

Fig. 20 shows the tumor growth curve of the CloudmanS91 homograft tumor model tumor-bearing mice after administration of 2E5. Data points represent mean tumor volumes within groups and error bars represent standard error (SEM).

Fig. 21 shows the survival curve of the CloudmanS91 homograft tumor model tumor-bearing mice administered with 2E5.

Detailed ways

The present invention will be further described below through specific embodiments and experimental data. Although specific terms are used hereinafter for purposes of clarity, these terms are not meant to define or limit the scope of the invention.

As used herein, the terms "programmed cell death 1", "programmed cell death 1", "protein PD-1", "PD-1", "PD1", "PDCD1", "hPD-1" and " hPD-F" are used interchangeably and include variants, isoforms, species homologues of human PD-1 and analogs having at least one common epitope of PD-1.

As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chains thereof. "Antibody" refers to a protein comprising at least two heavy (H) and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen.

The term "antibody", as used in this application, refers to an immunoglobulin or fragment or derivative thereof, and includes any polypeptide comprising an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, Mutated, grafted antibodies. The term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function, ie, are capable of specific binding to PD-1. Typically, such fragments will include antigen binding fragments.

The terms "antigen-binding fragment", "antigen-binding domain" and "binding fragment" refer to an antibody molecule comprising the amino acids responsible for the binding between a particular antibody and antigen. For example, where the antigen is large, the antigen-binding fragment only binds a portion of the antigen. That is, the part of the antigen molecule responsible for the specific interaction with the antigen-binding fragment is called "epitope" or "antigenic determinant".

An antigen-binding fragment typically includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not necessarily have to include both. For example, a so-called Fd antibody fragment consists of only the VH domain but still retains some of the antigen-binding functions of the intact antibody.

The above term "epitope" is defined as an antigenic determinant which specifically binds/recognizes the binding fragment. Binding fragments can specifically bind/react with conformational or continuous epitopes unique to the target structure, such as human PD-1 and murine PD-1 (mouse or rat). A conformational or discontinuous epitope is characterized by a polypeptide antigen as two or more discrete amino acid residues separated in the primary sequence, but aggregated together on the surface of the molecule when the polypeptide is folded into a native protein/antigen. An epitope is two or more discrete amino acid residues present on separate portions of one or more polypeptide chains. When the polypeptide chain folds into a three-dimensional structure, these residues gather on the surface of the molecule to form an epitope. In contrast, a continuous or linear epitope consisting of two or more discrete amino acid residues, which occurs in a single linear segment of a polypeptide chain.

The term "PD-1-binding epitope" refers to an antibody specifically binding to a specific epitope of PD-1, which can be defined by a linear amino acid sequence or a partial three-dimensional structure of PD-1. By binding is meant that the affinity of the antibody in the portion for PD-1 is significantly greater than its affinity for other related polypeptides. The term "substantially greater affinity" refers to a measurable increase in the affinity of a moiety for PD-1 compared to the affinity of other related polypeptides. Preferably, the affinity for a specific portion of PD- is at least 1.5 fold, 2 fold, 5 fold 10 fold, 100 fold, 10 ³ fold, 10 ⁴ fold, 10 ⁵ fold, 10 ⁶ fold or greater compared to other proteins. Preferably, binding affinity is determined by enzyme-linked immunosorbent assay (ELISA), or by fluorescence-activated cell sorting (FACS) analysis or surface plasmon resonance (SPR). More preferably, binding specificity is determined by fluorescence activated cell sorting (FACS) analysis.

The term "cross-reactivity" as described herein refers to the binding of antigenic fragments to the same human and murine target molecule. Thus, "cross-reactivity" should be understood as interspecies reactivity with the same molecule X expressed in a different species. The cross-reactivity specificity of monoclonal antibodies recognizing human PD-1, murine PD-1 (mouse or rat) can be determined by FACS analysis.

As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. Unless indicated otherwise, the terms "patient" or "subject" are used interchangeably.

The terms "treatment" and "method of treatment" refer to both therapeutic treatment and prophylactic/preventive measures. Those in need of treatment include those already with the particular medical condition, as well as those who may eventually acquire that condition.

The experimental methods in the following examples are conventional methods unless otherwise specified.

Example:

Embodiment 1 Experimental material preparation

1. Antigen Preparation

The DNA encoding the full length or extracellular region of PD-1 and PD-L1 was synthesized and inserted into the expression vector pcDNA3.3, respectively. After a large amount of plasmid DNA was extracted, the sequence of the inserted DNA fragment was verified by sequencing. The fusion protein PD-1 extracellular region and PD-L1 extracellular region contain different tags, including human Fc, mouse Fc and His tags, etc., by transfecting the PD-1 extracellular region gene sequence into CHO-S or HEK293 expressed in cells. Five days after the cells were transiently transfected, the cell culture supernatant was collected, and the fusion protein was purified and quantified for immunization and screening.

2. Establishment of Stable Cell Lines

In order to obtain antibody screening validation tools, PD-1 and PD-L1 transfected cell lines were prepared. Briefly, the pcDNA3.3 vector expression plasmid containing the full length of PD-1 or PD-L1 was transfected into CHO-K1 or 293F cells using Lipofectamine 2000 transfection reagent according to the experimental procedures provided by the manufacturer. After 48-72 hours of transfection, the transfected cells were cultured in a medium containing blasticidin or G418 to screen for cells inserted into the PD-1 or PD-L1 gene in the chromosome. At the same time, the cells were tested for the expression of PD-1 and PD-L1. Once expression was verified, single clones were picked by limiting dilution and expanded. The established monoclonal cell lines were then maintained in media containing lower doses of blasticidin or G418 antibiotics.

Example 2 Production of Antibody Hybridomas

1. Immunity

Female SD rats aged 6 to 8 weeks were each sensitized by plantar injection of 10 μg human PD-1 extracellular domain protein and 10 μg mouse PD-1 extracellular domain (in TiterMax) protein, and then used in Human PD-1 extracellular domain protein or mouse PD-1 extracellular domain protein in aluminum phosphate gel adjuvant was immunized once through the plantar until suitable for fusion. During the immunization period, the serum titer of anti-PD-1 antibody was detected every two weeks by ELISA or FACS method.

2. Cell Fusion

When the antibody titer reached a sufficiently high level, the rats were challenged with the final non-adjuvanted immunogen (human PD-1 extracellular domain protein and mouse PD-1 extracellular domain protein) (with an equal volume of phosphate buffered saline solution (PBS) instead of adjuvant). Resuscitate SP2/0 cells one week before fusion, subculture at a ratio of 1:2 to the day before fusion, and maintain the exponential growth of cells. On the day of fusion, take out the lymph nodes of SD rats under aseptic conditions, and process the lymph nodes into a single cell suspension as soon as possible, mix them with myeloma cells SP2/0 at a ratio of 1:1, treat them with protease solution, and use fetal bovine Serum was used to stop the reaction, and the original solution was replaced with ECF solution. The cell mixture was washed and resuspended with ECF solution, and the cell density in ECF was 2×10 ⁶ cells/ml. Immediately after electrofusion with a BTX 2000 electrofusion apparatus, the cell suspension was transferred from the fusion chamber to a sterile test tube containing more media, and incubated in a 37°C incubator for at least 24 hours. The cell suspension was then mixed and plated in a 96-well plate at a density of ¹ × 104 cells per well. The fused cells were cultured at 37°C, 5% CO2. After 7-14 days of colony culture, when the colony grows sufficiently large, transfer 100 μL supernatant from each well of the 96-well plate for antibody screening test.

3. First and second screening of hybridoma supernatants and competition confirmation

The ELISA method was used as a first-round screening method to test the binding of hybridoma supernatants to human PD-1 protein or mouse PD-1 protein. Briefly, ELISA plates (Nunc) were coated with 1 μg/mL human PD-1 extracellular domain protein or mouse PD-1 extracellular domain protein overnight at 4°C. After blocking and washing, the hybridoma supernatants were transferred to the coated microtiter plates and incubated at room temperature for 1 hour. The plate was then washed and then incubated with a goat anti-rat IgG Fc HRP (Bethyl) secondary antibody for 1 hour. After washing, TMB substrate was added for color development and the reaction was terminated with 2M HCl. The absorbance value at 450 nm was read using a microplate reader (Molecular Device).

To confirm the native binding of PD-1 antibodies to conformational PD-1 molecules expressed on the cell membrane, FACS analysis was performed on human PD-1-transfected CHO-S cell line or mouse PD-1-transfected 293F cell line as a second round of screening. CHO-S cells expressing human PD-1 or 293F cells expressing mouse PD-1 were transferred to a 96-well U-bottom plate (Corning) at a density of 1× ¹⁰⁵ cells per well, and then the hybridomas were plated The supernatant was transferred to the plate and incubated at 4°C for 1 hour. After washing with 1×PBS/1%BSA washing solution, goat anti-rat FITC secondary antibody (Jackson Immunoresearch Lab) was added and incubated with the cells at 4°C for 1 hour in the dark. Afterwards, the cells were washed and resuspended in 1×PBS/1% BSA or fixed in 4% formalin, and the results were analyzed by flow cytometry (BD) and FlowJo software. Binding of hybridoma supernatants to parental CHO-S cell line or 293F cell line was performed using the same method, respectively.

Antibody binding blocking activity against human PD-1/PD-L1 was tested as a confirmation screen to select potential target antibodies. By FACS analysis, the selected hybridoma supernatants were tested for their ability to block the binding of ligand PD-L1 to CHO-S cells transfected with human PD-1. CHO-S cells expressing human PD-1 were transferred to a 96-well U-bottom plate (Corning) at a density of 1×10 ⁵ cells per well. The hybridoma supernatants were then transferred to the plate and incubated at 4°C for 1 hour. After washing with 1×PBS/1% BSA, add mouse Fc-fused human PD-L1 extracellular domain protein or mouse Fc-fused mouse PD-L1 extracellular domain protein and incubate at 4°C for 1 Hour. After washing, goat anti-mouse Fc FITC secondary antibody (no cross-reactivity with rat IgG Fc, Jackson Immunoresearch Lab) was added and incubated with cells at 4°C in the dark for 1 hour. Afterwards, the cells were washed and resuspended in 1×PBS/1% BSA or fixed in 4% formalin, and the results were analyzed by flow cytometry (BD) and FlowJo software.

Figure 1 shows the binding of 16 hybridoma antibodies to human PD-1 or mouse PD-1 on the cell surface, Figure 1A shows the binding of 16 hybridoma antibodies to human PD-1 on the cell surface; Figure 1B shows the binding of the hybridoma antibodies Binding to mouse PD-1 on the cell surface.

4. Hybridoma Subcloning

Once specific binding and blocking are verified by first-round, second-round and competition confirmation screens, positive hybridoma cell lines are picked for subcloning. Briefly, for each hybridoma cell line, cells were counted and diluted to 5 cells per well, 1 cell per well and 0.5 cells per well in cloning medium. Add 200 μL of diluted cloning medium to each well of a 96-well plate, with 5 cells per well for one plate, 1 cell per well for one plate, and 0.5 cells per well for four plates. All plates were cultured at 37°C and 5% CO ₂ until all cells could be detected by ELISA or FACS. The detection method is the same as above, and the positive monoclonals are selected for expansion culture, and the purified antibodies are subjected to further characterization analysis.

5. Subtype testing

50 μL per well of goat anti-rat IgG1, IgG2a, IgG2b, IgG2c, IgG or IgM antibody at a concentration of 1 μg/mL were coated on the microtiter plate (Nunc) overnight. After blocking, 50 μL of hybridoma supernatant samples were added to each well and incubated at room temperature for 2 hours. Secondary goat anti-rat IgG kappa or lambda light chain-HRP (Bethyl) antibodies were used as detection antibodies. Color development was performed using TMB substrate, and the reaction was terminated with 2M HCl. The absorbance value at 450nM was read with a microplate reader (Molecular Device).

Table 3 shows the subtype results of 16 hybridoma antibodies, among which 7 antibodies are polyclonal, and the remaining 9 antibodies are IgG2a kappa subtype. Considering that the anti-PD-1 antibody needs to avoid ADCC and CDC in vivo, the antibody was constructed as a human IgG4kappa subtype after humanization.

Table 3 Subtypes of hybridoma antibodies

Example 3 Antibody Hybridoma Cell Sequencing, Antibody Humanization Construction and Affinity Maturation

1. Antibody sequencing of hybridoma cells

Monoclonal hybridoma RNA was isolated using Trizol reagent. The VH and VL segments of the PD-1 chimeric antibody are amplified by the following method: first, the RNA is reverse-transcribed into cDNA by using reverse transcriptase,

Reaction system (20μL)

Reaction conditions

first step second step third step the fourth step temperature(°C) 25 37 85 4 time 10 minutes 120 minutes 5 ∞

The resulting cDNA was used as a template for the following PCR amplification using specific primers for the gene of interest. The PCR reaction operation is as follows:

Reaction conditions:

The resulting PCR reaction product (10 μL) was ligated to the pMD18-T vector. 10 μL of the ligation product was transformed into Top10 competent cells. Using M13-48 and M13-47 primers, PCR was used to verify positive clones and then sequenced.

2. Construction of humanized antibody molecules

According to the high affinity and specificity of binding to PD-1, the rat anti-human PD-1 antibody from the hybridoma is selected for humanization, which is used to improve the homology degree of the antibody sequence derived from the rat and the antibody sequence of the human . The humanization is performed using a technique known as CDR grafting. The KABAT system and the IMGT system were used to divide the FR regions and CDR regions of antibody variable region genes. In the antibody database, combined with the comparison results of sequence homology and structural similarity, select the FR1-3 region gene of the antibody variable region of similar human origin to replace the FR1-3 region gene of mouse origin, and select the gene with the best structure. The similar human JH and JK genes replace the murine FR4 gene. After verifying the template sequence and optimizing codons, the heavy and light chain variable regions were amplified and cloned into expression vectors to express humanized antibodies.

According to the binding ability of hybridoma antibody to human and mouse PD-1 protein, two antibodies W3052_r16.88.9 and W3052_r16.81.3 were selected for humanization. After humanization transformation, the humanized antibody W3052_r16.88 derived from the parental hybridoma antibody W3052_r16.88.9 was selected based on the degree of humanization of different antibodies and the strength of binding ability to human and mouse PD-1 proteins -z9-IgG4(42720) was affinity matured (Table 4).

Table 4

3. Affinity maturity

Each amino acid in the heavy chain CDR3 region, light chain CDR1 region and CDR3 region of the humanized antibody is mutated to other 20 amino acids by hybrid mutation method. Mutations were introduced at the position of each of the targeted CDRs using DNA primers containing NNS codons encoding 20 amino acids. Use a single degenerate primer in the hybridization mutagenesis reaction. Briefly, each degenerate primer was phosphorylated and then used in a 10:1 ratio with uridinylated ssDNA. The mixture was heated to 85°C for 5 minutes and then cooled to 55°C over 1 hour. Thereafter, T4 ligase and T4 DNA polymerase were added, and the mixture was incubated at 37°C for 1.5 hours. Synthetic products of the CDRs of VH and VL, combined separately. Typically, 200ng of pooled library DNA was electrotransformed into BL21 to form BL21 lawns or plaques producing scFv fragments.

The primary screen consisted of a single point ELISA (SPE) assay using periplasmic extracts (PE) of bacteria grown in 96-well plates (deep wells). Briefly, the capture ELISA involved coating wells of a 96-well Maxisorp immunoplate overnight at 4°C with anti-c-myc antibody in pH 9.2 coating buffer (200 mM sodium carbonate/bicarbonate). The next day, the plates were blocked with casein for 1 hour at room temperature. The PE of the scFv was then added to the plate and incubated for 1 hour at room temperature. After washing, biotinylated antigenic protein was added to the wells, and the mixture was incubated at room temperature for 1 hour. This was followed by incubation with streptavidin-HRP conjugate for 1 hour at room temperature. HRP activity was detected with TMB substrate and the reaction was terminated with 2M hydrochloric acid. The absorbance value at 450 nm was read with a microplate reader (Molecular Device). The clones whose absorbance value at 450nm was higher than that of the parental antibody were picked and tested again by ELISA for confirmation, and the result was positive. Clones that repeatedly exhibited a greater signal than the parental antibody were sequenced. The scFv protein concentration of clones with CDR alterations was then determined by a quantitative scFv ELISA method using a known concentration of scFv as a reference. The scFv protein concentration was determined by comparing the ELISA signal with that produced by a reference scFv. To determine the relative binding affinity of the mutated scFv to the parental antibody, the binding assay was repeated for all positive variants at normalized scFv concentrations.

Point mutations of VH and VL determined to be beneficial for antigen binding are further combined to obtain additional binding synergy. The combinatorial mutants were expressed as scFv and screened using capture ELISA. Clones with higher absorbance values than the parent antibody were selected for sequencing and their affinity was further determined by ELISA method.

Figure 2 is the result of the first round of mutant library screening. After the second round of screening for affinity maturation, 10 humanized antibodies including 2E5, 2G4, 1G10, 2C2, 2B1, 8C10, 1H6, 5C4, A6W and L1I were obtained, and their affinity data and specific The CDR sequences are shown in Table 5.

Table 5 is the results of the second round of mutant library screening. Among them, the affinity results of these antibodies to human, cynomolgus monkey and mouse PD-1 were combined, and four antibodies 1H6, 2E5, 2G4 and 2C2 were selected for further characterization.

table 5

4. Antibody Purification

293F cells were transfected with DNA vectors containing affinity-matured humanized antibodies for antibody expression and production. Antibodies in 293F cell culture supernatants were purified using protein A affinity chromatography columns.

The characterization of embodiment 4 humanized antibody

1. Binding experiment with human, mouse and cynomolgus monkey PD-1

1.1 Binding experiment determined by FACS

In order to test the binding ability of the antibody to the cell surface PD-1 protein, different concentrations of the antibody were mixed with CHO-S cells expressing human PD-1 or 293F cells expressing mouse PD-1 or activated cynomolgus monkey PBMC at 4°C. Incubate for 1 hour under conditions. After washing, antibody binding to cells was detected using FITC-labeled goat anti-human IgG Fc secondary antibody (Jackson Immunoresearch Lab). The results were then analyzed using flow cytometry (BD) and FlowJo software. For specific experimental steps, see Part 3 of Example 2.

Figure 3A shows the binding curve of the humanized antibody to human PD-1 on the cell surface, and the antibody specifically binds to human PD-1 with an EC50 of 2.20-2.78nM. Figure 3B shows the binding curve of the humanized antibody to mouse PD-1 on the cell surface, and the antibody specifically binds to mouse PD-1 with an EC50 of 11.8-15.1 nM. Figure 3C shows that the binding of humanized antibody to activated cynomolgus monkey PBMC has a dose-dependent relationship. The isotype control is human IgG4kappa. The same below.

1.2 Species cross-reaction test with human, mouse and cynomolgus monkey PD-1

The cross-reactivity of antibodies to cynomolgus monkey and mouse PD-1 proteins was determined by ELISA method. 1 μg/mL human, cynomolgus monkey and mouse PD-1 extracellular region proteins (Sino Bioligical) were coated on the microtiter plate (Nunc) overnight at 4°C. After blocking, humanized antibodies were added to the plate and incubated for 1 hour at room temperature. Binding of the antibody to the coated antigen was detected using goat anti-human IgG Fc-HRP (Bethyl) as the secondary antibody. Color development was performed using TMB substrate and the reaction was stopped with 2M HCl. The absorbance value at 450 nm was read with a microplate reader (Molecular Device).

Figure 4 shows the results of the ELISA of the species cross-reactivity test between the antibody and human, mouse, and cynomolgus monkey PD-1. Dependent form binding. Figure 4A is the binding of humanized PD-1 antibody to human PD-1 protein; Figure 4B is the binding of humanized PD-1 antibody to mouse PD-1 protein; Figure 4C is the binding of humanized PD-1 antibody to human PD-1 protein Cynomolgus monkey PD-1 protein binding

2 Cross-reaction test with PD-1 family CD28 and CTLA4

The cross-reactivity of the humanized antibody with CD28 and CTLA-4 proteins of the same family as PD-1 was detected by FACS method. In short, the constructed CHO-S cells expressing human PD-1, CHO-K1 cells expressing human CD28 or 293F cells expressing human CTLA-4 were seeded in 96-well U-bottom plates (BD), The cell density was ² x 105 cells per well. Dilute test antibody into washing solution (1×PBS/1%BSA) and incubate with CHO-S cells expressing human PD-1, CHO-K1 cells expressing human CD28 or 293F cells expressing human CTLA-4 at 4°C Incubate for 1 hour respectively. After washing the cells, FITC-labeled goat anti-human IgG Fc (Jackson Immunoresearch Lab) secondary antibody was added, and incubated at 4°C in the dark for 1 hour. Then the cells were washed once, and the cells were resuspended with 1×PBS/1%BSA, and the results were analyzed by flow cytometry (BD) and FlowJo software.

Figure 5 shows the results of cross-reactivity of humanized antibodies with CD28 and CTLA-4 proteins of the same family as PD-1. The results showed that the antibody specifically bound to PD-1, but not to CD28 and CTLA-4 of the same family as PD-1.

3. Competition experiment

3.1 Use FACS to detect the ability of PD-1 antibody to block PD-L1 binding to PD-1

In order to test whether the humanized antibody can block the combination of PD-L1 and PD-1, CHO-S cells expressing human PD-1 or 293F cells expressing mouse PD-1 were mixed with different concentrations of antibody at 4°C. Incubate for 1 hour. Unbound antibodies were washed away, and mouse Fc-labeled human or mouse PD-L1 proteins were added, respectively. After incubation at 4°C for 1 hour, the binding of ligand PD-L1 to PD-1-expressing cells was detected using FITC-labeled goat anti-mouse IgG Fc secondary antibody (Jackson Immunoresearch Lab), followed by flow cytometry (BD) and FlowJo software was used to analyze the results.

3.2 ELISA method is used to detect whether the humanized antibody can block the binding of PD-L2 and PD-1

Briefly, ELISA plates (Nunc) were coated with 1 μg/ml human PD-1 extracellular domain protein overnight at 4°C. After blocking and washing, humanized antibodies diluted in different concentrations were pre-mixed with a constant concentration of His-tagged PD-L2 extracellular domain protein, added to the coated microtiter plate and incubated at room temperature for 1 hour. Afterwards, the microtiter plate was washed and then a goat anti-HisHRP (GenScript) secondary antibody was added to incubate for 1 hour. After washing, TMB substrate was added for color development and the reaction was terminated with 2M HCl. The absorbance value at 450 nm was read using a microplate reader (Molecular Device).

Figure 6A shows that a humanized antibody blocks the binding of human PD-L1 to human PD-1 on the surface of CHO-S cells, and Figure 6B shows that a humanized antibody blocks the binding of mouse PD-L1 to the surface of 293F cells. Binding of PD-1. Figure 7 shows that the humanized antibody blocks the binding of human PD-L2 to PD-1 protein, and the blocking effect is dose-dependent.

4. Affinity test determined by surface plasmon resonance (SPR)

The affinity and binding kinetics of the antibody to PD-1 were characterized by SPR method using ProteOn XPR36 (Bio-Rad). Protein A protein (Sigma) was immobilized on a GLM sensor chip (Bio-Rad) by amine coupling. Purified antibodies are flowed over the sensor chip and captured by protein A. The chip was rotated 90° and washed with electrophoresis buffer (1×PBS/0.01% Tween20, Bio-Rad) until the baseline stabilized. 7 concentrations of human PD-1 protein and electrophoresis buffer flowed through the antibody flow cell at a flow rate of 30 μL/min, first the binding phase flowed for 180 s, and then the dissociation phase flowed for 300 s. _The chip was regenerated with H3PO4 at pH 1.5 after each run _. Association and dissociation curves were fitted to a 1:1 Langmiur binding model using ProteOn software. The affinity test method for antibody and mouse PD-1 protein is the same as above.

Table 6 shows the results of the surface plasmon resonance detection of the affinity of the humanized PD-1 antibody to recombinant human or recombinant mouse PD-1. The control antibody 1 (WBP305BMK1) was synthesized according to the 5C4 sequence in BMS patent US9084776B2, that is, the anti-PD-1 drug Opdivo that BMS company has listed; the control antibody 2 (Keytruda) is the anti-PD-1 drug Keytruda that Merck company has listed. The same below. As shown in Table 6A, the affinity of the humanized PD-1 antibody to recombinant human PD-1 detected by using surface plasmon resonance was from 1.43E-8 to 5.64E-9 mol/L. Compared with WBP305BMK1 and Keytruda, the K _D value of the antibody in this application is smaller, indicating that 2E5, 2G4, and 2C2 have better binding ability to human PD-1. As shown in Table 6B, the affinity of the humanized PD-1 antibody to recombinant mouse PD-1 detected by using surface plasmon resonance was from 9.37E-9 to 3.89E-9 mol/L.

Table 6A

Table 6B

5. FACS determination of the affinity test of anti-PD-1 antibody and PD-1 molecules on the cell surface

CHO-S cells expressing human PD-1 or 293F cells expressing mouse PD-1 were seeded in a 96-well U-bottom plate (BD) at a density of ¹ ×105 cells per well. Test antibodies were serially diluted 1:2 in wash solution (1×PBS/1% BSA) and incubated with cells for 1 hour at 4°C. Goat anti-human IgG Fc-FITC secondary antibody (3.0 moles FITC per mole of IgG, Jackson Immunoresearch Lab) was added and incubated at 4°C in the dark for 1 hour. Cells were then washed once and resuspended in 1×PBS/1% BSA and analyzed using flow cytometry (BD). Based on quantitative beads Quantum ^TM MESF Kit (Bangs Laboratories, Inc.), the fluorescence intensity will be converted to correlate with molecules/cells. KD was calculated using Graphpad _Prism5 .

As shown in Table 7A-7B, the affinity of the humanized PD-1 antibody to the human PD-1 on the surface of CHO-S cells detected by using the FACS method shows that the humanized PD-1 antibody has an affinity for human PD-1 on the surface of CHO-S cells. The affinity of PD-1 ranges from 3.80E-10 to 2.15E-10mol/L. The affinity of the humanized PD-1 antibody to mouse PD-1 on the surface of 293F cells ranges from 5.39E-08 to 1.74E-08mol/L.

Table 7A

Table 7B

6. Epitope test

FACS determination of antigenic epitope competition test: This experiment is mainly to find out whether the antibodies bind to the same, similar or completely different antigenic epitopes. To check whether the humanized antibody binds to the same epitope as the control antibody, CHO-S cells expressing human PD-1 were mixed with the test antibody (serially diluted with washing buffer) and biotin-labeled control antibody A or B ( 1 μg/mL) and incubated at 4°C for 1 hour. Cells were washed, PE-conjugated streptavidin was added as a secondary antibody, and incubated at 4°C for 30 min. Cells were washed once and resuspended with 1×PBS/1% BSA, followed by flow cytometry (BD) and FlowJo software for result analysis.

The results of Figures 8A-8B show the epitope test results show that the humanized PD-1 antibody binds to the same or similar epitope as the control antibody. Figure 8A shows epitopes competing with control antibody 1 (WBP305BMK1 ), and Figure 8B shows epitopes competing with control antibody 2 (Keytruda).

In addition, an alanine scanning experiment was further performed on human PD-1 (hPD-1) to evaluate its effect on antibody binding. The alanine residue in hPD-1 was mutated to a glycine codon and the remaining residues were mutated to alanine. Point mutation substitutions were performed on each residue of hPD-1 extracellular domain by two-step serial PCR method. In the first step of PCR, the pcDNA3.3-hPD-1_ECD.His plasmid containing the hPD-1 ectodomain and the C-terminal His tag coding sequence was used as a template, and the QuikChangelightning multiple point mutation kit (Agilent technologies, PaloAlto, CA) was used and Mutation primers. After the mutant strand synthesis reaction, the master was digested with DpnI endonuclease. The second step PCR amplified linear DNA containing CMV promoter, PD-1 extracellular domain (ECD), His tag and herpes simplex virus thymidine kinase (TK) polyadenylation and expressed it in HEK293F Transient expression in cells (Life Technologies, Gaithersburg, MD).

Immunoenzyme-linked adsorption (ELISA) binding assay was performed with monoclonal antibody W3052_r16.88.9 and Keytruda-coated plates. After binding to the supernatant containing quantitative PD-1 mutant or human/mouse His-tagged PD-1 extracellular domain protein (SinoBiological, China), an HRP-coupled anti-His antibody was added as a detection antibody. Absorbance was normalized to the mean absorbance of control mutants. After setting an additional cutoff (<0.55) for the fold change in binding, the epitope residues of the final determinants were found.

The binding effect of antibody W3052_r16.88.9 and Keytruda on human and mouse PD-1 was tested (Figure 9). Our first antibody W3052_r16.88.9 was found to bind both hPD-1 and murine PD-1 (mPD-1), while Keytruda only bound hPD-1 (Figure 9). The unique functional cross-reactivity of W3052_r16.88.9 can help to provide more animal model options in preclinical studies of drug safety evaluation. To investigate the reason for the observed binding behavior described above, we performed epitope mapping.

Table 8 shows that hPD-1 mutants with 30 point substitutions significantly reduced binding to antibodies. By checking the positions of all these residues on the hPD-1 crystal structure (PDB code 3RRQ and 4ZQK), it was found that some amino acids (such as Val144, Leu142, Val110, Met108, Cys123, etc.) were completely embedded in the protein, which is impossible Make direct contact with the antibody. The found decreased binding is most likely caused by the structural instability or even structural collapse of hPD-1 after alanine substitution. According to antigen structure analysis, some residues are not involved in binding, but are expected to respond to the structural stability of hPD-1, such as V144 and L142. Additionally, mutations that could affect both antibodies simultaneously were considered false hotspots and were removed from the list. After setting an additional cutoff (<0.55) for the fold change in binding, the final identified antigenic residues are listed in Table 9. Among them, 9 locations correspond to W3052_r16.88.9, and 5 correspond to Keytruda.

The antigen amino acid comparison of W3052_r16.88.9 and Keytruda in Table 9 shows that only two residue hotspots overlap, and the rest seem very scattered, indicating that the two antibodies use different mechanisms for hPD-1 binding and hPD-L1 blocking. Reading the residue IDs in Figure 8 does not directly elucidate the mechanism. Therefore, for better display and comparison, all the data and hPD-L1 binding sites in Table 9 have been mapped and compared in the hPD-1 crystal structure (Figure 10)

Table 8. Effect of PD-1 point mutations on antibody binding

^a The fold change in binding is the relative value of multiple alanine silent substitutions

Table 9. Discovery of potential epitopes

Critical value: multiple of change <0.55

^* The C" strand observed in mPD-1 is not present in the hPD-1 structure. This β-sheet structure is replaced by an unstructured loop region in hPD-1. For easier comparison with mPD-1 Yes, we still mark the area with a C".

Although both have hPD-1 binding and hPD-L1 blocking functions, the two studied antibodies W3052_r16.88.9 and Keytruda have significantly different epitopes (Fig. 10B, 10C). The Keytruda epitope is mainly contributed by the C'D loop region (corresponding to the mPD-1 C" chain), which is completely disjoint with the PD-L1 binding site. This suggests that the hPD-L1 blocking function of Keytruda is more dependent on its antibody size The steric hindrance effect. Relatively speaking, the surface map results showed that the epitope of W3052_r16.88.9 consisted of hotspots distributed in multiple regions and directly overlapped with the binding site of hPD-L1 (Fig. 10A, 10B). W3052_r16. 88.9 blocks hPD-L1 by competing with hPD-L1 for its consensus binding site. In addition, W3052_r16.88.9 does not interact with the C'D flexible loop region (or the corresponding C" strand of mPD-1), which is in the human Large structural deviations were shown in PD-1 of human and murine origin (Figure 11). Its main action point is on the FG loop region (Lin et al. (2008) PNAS 105:3011-3016). This explains why W3052_r16.88.9 can bind PD-1 from both sources, while Keytruda can only bind human PD-1 (Figure 9). Due to this unique functional cross-reactivity, the preclinical safety assessment of W3052_r16.88.9 can be carried out in mouse models, which greatly simplifies and accelerates its development process. All in all, W3052_r16.88.9 is predicted to be more functional and exploitable than Keytruda.

7. Determination of the in vitro function of PD-1 antibody by cell experiments

To assess the ability of humanized antibodies to modulate T cell responses, including cytokine production and cell proliferation, the following three experiments were performed using affinity matured humanized PD-1 antibodies as well as a control antibody.

7.1 Allogeneic mixed lymphocyte reaction MLR is used to detect the effect of antibodies on T cell function

Isolation of human DC cells, CD4 ⁺ T cells, CD8 ⁺ T cells and total T cells: Human PBMC cells were freshly isolated from healthy donors using Ficoll-Paque PLUS (GE) gradient centrifugation. Monocytes were isolated from healthy donors using the Human Monocyte Enrichment Kit (StemCell) according to the instructions. Cells were cultured for 5-7 days in a medium containing rhGM-CSF and rhIL-4 to induce dendritic cells (DC). 18 to 24 hours before MLR, 1 μg/mL LPS was added to the medium to induce maturation of DC cells. Human CD4 ⁺ T cells were isolated using the human CD4 ⁺ T cell enrichment kit (StemCell) according to the instructions. Using the mouse CD4 ⁺ T cell enrichment kit (StemCell), mouse CD4 ⁺ T cells were isolated from the spleen of Balb/c mice according to the instructions. Bone marrow cells from C57BL/6 mice were induced into DC cells by culture medium containing rmGM-CSF and rmIL-4 for 5-7 days. 18 to 24 hours before MLR, 1 μg/mL LPS was added to the medium to induce maturation of DC cells.

Briefly, primary dendritic cell (DC) stimulation of MLR was performed in 96-well U-bottom tissue culture plates in 200 microliters of RPMI1640 containing 10% FCS and 1% antibiotics. In the presence or absence of test antibody or baseline antibody, DC cells were mixed with ¹ ×105 CD4 ⁺ T cells, the ratio of DC cells to T cells was between 1:10 and 1:200 (from 166.75nM to 0.00667nM, usually a total of six concentrations). To determine the effect of anti-PD-1 on T cell function, cytokine production and T cell proliferation were measured. Results shown are representative of at least five experiments performed.

Factor detection : Human IFN-γ and IL-2 were measured by enzyme-linked immunosorbent assay (ELISA) using matched antibodies. Plates were pre-coated with capture antibodies specific for human IFN-γ (cat#Pierce-M700A) or IL-2 (cat#R&D-MAB602), respectively. Biotin-conjugated anti-IFN-γ antibody (cat#Pierce-M701B) or anti-IL-2 antibody (cat#R&D-BAF202) was used as detection antibody.

As shown in Figure 12A, all tested anti-PD-1 antibodies increased IL-2 secretion in a dose-dependent manner. Figure 12B shows that anti-PD-1 antibodies increased IFN-γ secretion in a dose-dependent manner.

Proliferation experiment : 3H thymidine (cat#PerkinElmer-NET027001MC) was diluted 1:20 with 0.9% NaCl solution, and added to the cell culture plate at 0.5 uCi per well. Plates were incubated at 37°C for 16 to 18 hours in 5% CO ₂ prior to assay of 3H-thymidine incorporation into proliferating cells. As shown in Figure 12C, all tested anti-PD-1 antibodies increased the proliferation of T cells in a dose-dependent manner.

In order to detect the effect of the humanized anti-PD-1 antibody on the proliferation of mouse T cells in the MLR, the effects of the humanized antibody on the production of mouse IL-2 and IFN-γ in the MLR and the proliferation of mouse T cells The function detection method is the same as above. As shown in Figure 13A, all tested anti-PD-1 antibodies increased IL-2 secretion in a dose-dependent manner. Figure 13B shows that anti-PD-1 antibodies increased IFN-γ secretion in a dose-dependent manner. As shown in Figure 13C, all tested anti-PD-1 antibodies increased the proliferation of T cells in a dose-dependent manner.

7.2 Effect of PD-1 antibody on cell proliferation and factor production under autoantigen-specific immune response

CD4 ⁺ T cells and DC cells were isolated from the same CMV+ donor. Briefly, CD4 ⁺ T cells were purified from PBMCs and cultured in the presence of CMVpp65 polypeptide and a low dose of IL-2 (20 U/mL). At the same time, DCs were obtained by culturing monocytes according to the aforementioned method. After 5 days, pp65 polypeptide was added to DC cells for pre-incubation at 37° C. for 1 hour, and then DC was added to CD4 ⁺ T cells in the presence or absence of humanized antibody or control antibody. On the 5th day, the IFN-γ level in the culture supernatant was measured by ELISA method. Proliferation of CMVpp65-specific CD4 ⁺ T cells was determined by 3H thymidine incorporation as previously described.

14A-14B show the results of human allogeneic mixed lymphocyte reaction (MLR), demonstrating that PD-1 antibody can enhance the function of human CD4 ⁺ T cells. Figure 14A shows that humanized PD-1 antibodies increase IFN-γ production in specific T cell responses. Figure 14B shows that humanized PD-1 antibody increases concentration-dependent proliferation of CMV+CD4 ⁺ T cells loaded with autologous DCs loaded with CMVpp65 polypeptide.

7.3 Inhibitory function of human anti-PD-1 antibody on regulatory T cells ( Treg)

Regulatory T cells (Treg), a subgroup of T cells, are key immune regulators and play an important role in maintaining self-tolerance. CD4+CD25+ Treg cells are tumor-associated, as increased Treg numbers are found in patients with multiple cancers and are associated with poorer prognosis. To directly assess the role of PD-1 humanized antibodies in inhibiting Treg suppressive function, Treg function was compared in the presence or absence of humanized or control antibodies. Briefly, CD4+CD25+Treg cells and CD4+CD25-T cells were isolated by anti-CD25 specific micro-magnetic beads (StemCell) method and product instructions, and 2000 DC cells, ¹ ×105 CD4+CD25+Treg Cells were co-cultured with 1×10 ⁵ CD4+CD25-T cells and PD-1 antibody in a 96-well plate. Plates were co-cultured for 5 days at 37°C, 5% CO ₂ . IFN-γ cytokine production and T cell proliferation were measured as previously described.

Figure 15 demonstrates that PD-1 antibody can reverse the suppressive function of Treg. Figure 15A shows that PD-1 antibody restored IFN-γ secretion. Figure 15A shows that PD-1 antibody restores the proliferation of effector T cells

8. ADCC/CDC test

Since human PD-1 is expressed in multiple cell types, in order to minimize unwanted toxicity to healthy PD-1 positive immune cells, it was verified that the selected humanized PD-1 antibodies do not have ADCC and CDC functions.

8.1 ADCC detection

Target cells (activated CD4+ T cells) and different concentrations of humanized antibodies were pre-incubated in a 96-well plate for 30 minutes, and then PBMC (effector cells) were added at a ratio of effector cells/target cells of 50:1. The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 6 hours. Lysis of target cells was measured by the Cyto-LDH Toxicity Assay Kit (Roche). The absorbance value at 492 nm was read using a microplate reader (Molecular Device). Herceptin (Roche) and human breast cancer cell line SK-Br-3 (HER2 positive) were used as positive controls.

Figure 16 shows that humanized PD-1 antibody does not mediate ADCC using PBMC as a source of natural killer (NK) cells and activated CD4 ⁺ T cells expressing high levels of PD-1 as target cells.

8.2 CDC detection

Target cells (activated CD4 ⁺ T cells), diluted human serum complement (Quidel-A112) and different concentrations of humanized antibodies were mixed in a 96-well plate. The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 4 hours. Target cell lysis was measured using CellTiterGlo (Promega-G7573). Rituximab (Roche) and the human CD20 positive cell line Ramos served as positive controls.

Figure 17 shows target cells (activated CD4+ T cells), diluted human serum complement (Quidel-A112) and different concentrations of humanized PD-1 antibody were incubated for 4 hours, using CellTiterGlo (Promega-G7573) to determine Lysis of target cells. The data show that humanized PD-1 antibody does not mediate CDC effect.

Example 5 Treatment of Human PD-1 Monoclonal Antibody in In Vivo Tumor Model

1. Experimental Design

Table 10. Animal grouping and dosing regimen for 2E5 in vivo efficacy experiments

Note:

1.N: the number of mice in each group

2. Administration volume: 10 μL/g based on mouse body weight. If body weight is lost by more than 15%, the dosing regimen should be adjusted accordingly.

2. Experimental method and steps

2.1 Cell culture

Cell culture: Murine melanoma CloudmanS91 cells (ATCC-CCL-53.1) were cultured in vitro as a single layer. The culture conditions were F-12K medium plus 2.5% fetal bovine serum and 15% horse serum, 100 U/mL penicillin and 100 μg/mL chain Mycin, cultured at 37°C, 5% CO ₂ . Routine digestion with trypsin-EDTA was performed twice a week for passaging. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected, counted, and inoculated.

2.2 Tumor cell inoculation

0.1 mL (5×10 ⁵ cells) of Cloudman S91 cells were subcutaneously inoculated on the right back of each mouse, and the administration began when the average tumor volume reached about 64 mm ³ . The experimental grouping and dosing regimen are shown in Table 10.

2.3 Tumor measurement and experimental indicators

The experimental index is to investigate whether tumor growth is inhibited, delayed or cured. Tumor diameters were measured with vernier calipers three times a week. The formula for calculating the tumor volume is: V=0.5a×b ² , where a and b represent the long diameter and short diameter of the tumor, respectively.

The antitumor efficacy of compounds was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). TGI (%) reflects tumor growth inhibition rate. Calculation of TGI (%): TGI (%)=[(1-(average tumor volume at the end of administration of a treatment group-average tumor volume at the beginning of administration of this treatment group))/(average tumor volume at the end of treatment of the solvent control group Volume-vehicle control group (average tumor volume at the beginning of treatment)]×100%.

Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C%=T _RTV /C _RTV ×100% (T _RTV : RTV of the treatment group; C _RTV : RTV of the negative control group). The relative tumor volume (RTV) was calculated according to the results of tumor measurement, and the calculation formula was RTV=Vt/V0, where V0 was the average tumor volume measured during group administration (i.e. d0), and Vt was the value of a certain measurement The average tumor volume, T _RTV and C _RTV take the data of the same day.

TC (days) reflects the tumor growth delay index, T represents the average number of days used for the tumors in the treatment group to reach a preset volume (such as 300mm ³ ), and C represents the average days used for the tumors in the control group to reach the same volume.

The survival curve was drawn, and the animal survival time was defined as the time from drug administration to animal death or from drug administration to the time when the tumor volume reached 2000 mm ³ , and the animal was deemed dead if one of them was satisfied. The median survival time (days) of animals in each group was calculated. By comparing the median survival of the treatment group and the model control group, the increase in survival (ILS) was calculated, expressed as a percentage of survival over the model control group.

2.4 Statistical analysis

Statistical analysis, including mean and standard error (SEM) of tumor volume at each time point of each group (see Table 11 for specific data). The whole experiment ended 37 days after the administration, and the animals in each group were euthanized successively on the 13th day after the administration. Therefore, the tumor volume on the 13th day after the start of the administration was used for statistical analysis to evaluate the differences between the groups. The comparison between two groups was analyzed by T-test, and the comparison between three or more groups was analyzed by one-way ANOVA. If the F value was significantly different, the Games-Howell method was used for testing. If there is no significant difference in the F value, the Dunnet (2-sided) method is used for analysis. All data analyzes were performed with SPSS 17.0. p<0.05 considered significant difference. The survival time of animals was analyzed by Kaplan-Meier method Log-rank test.

3. Experimental results

3.1 Mortality, morbidity and body weight changes

The body weight of experimental animals was used as a reference index for indirect determination of drug toxicity. Figure 18 and Figure 19 show the effect of 2E5 on the body weight of CloudmanS91 cell subcutaneous syngeneic transplantation tumor female DBA/2 mouse model. None of the dosing groups showed significant body weight loss in this model (Figure 18). Therefore, 2E5 has no obvious toxicity in the mouse melanoma CloudmanS91 model.

3.2 Tumor volume

Table 11 shows the changes in tumor volume in each group after treatment with CloudmanS91 cell subcutaneous syngeneic transplantation tumor female DBA/2 mouse model 2E5.

Table 11. Tumor volume of each group at different time points

Note:

a. Mean ± SEM,

b. Days after administration.

3.3 Tumor growth curve

Tumor growth curves are shown in Figure 20.

3.4 Anti-tumor efficacy evaluation index

Table 12. Evaluation of antitumor efficacy of 2E5 on the CloudmanS91 syngeneic tumor model (calculated based on the tumor volume on the 13th day after administration)

Note:

a. Mean ± SEM.

b. Tumor growth inhibition was calculated from T/C and TGI (TGI (%)=[1-(T13-T0)/(V13-V0)]×100).

c. p-values are calculated based on tumor volume.

3.5 Survival curve

The survival curves of animals in each group are shown in Figure 21.

3.6 Survival time

Table 13. The effect of 2E5 on the survival time of CloudmanS91 homologous tumor model animals

Note: a.p value represents the comparison between each administration group and the vehicle control group.

b. At the end of the experiment, the survival rate of animals in the 2E53mg/kg group was 66.7%.

4. Experimental results and discussion

In this experiment, we evaluated the in vivo efficacy of 2E5 in the CloudmanS91 syngeneic tumor model. The tumor volumes of each group at different time points are shown in Table 11, Table 12 and Figure 20, and the survival period is shown in Figure 21 and Table 13. Thirteen days after the start of administration, the tumor volume of the tumor-bearing mice in the solvent control group reached 1,626 mm ³ . Compared with the solvent control group, the 1mg/kg group of the test substance 2E5 had a weak tumor inhibitory effect, the tumor volume was 1,089mm ³ (T/C=68.1%, TGI=34.4%, p=0.367), and the days of tumor growth delay is 0 days. Compared with the solvent control group, the 3mg/kg group of 2E5 had a significant tumor-inhibiting effect, the tumor volume was 361mm ³ (T/C=22.9%, TGI=81.0%, p=0.008), and the days of tumor growth delay were 5 days. Compared with the solvent control group, the 10mg/kg group of 2E5 also had a significant tumor inhibitory effect, with a tumor volume of 614mm ³ (T/C=39.4%, TGI=64.7%, p=0.036), and the number of days of tumor growth delay was 5 days .

During the whole experiment, the median survival period of the tumor-bearing mice in the solvent control group was 16 days. Compared with the vehicle control group, the median survival period of the tumor-bearing mice in the 1mg/kg group of test substance 2E5 was 20 days, and the survival period was prolonged by 25% (p=0.077); The survival rate of tumor mice was 66.7% (p=0.001). The median survival period of tumor-bearing mice in the 10 mg/kg group of test substance 2E5 was 32 days, and the survival period was prolonged by 100% (p=0.022).

The effect of the 2E5 test substance on the body weight change of tumor-bearing mice is shown in Figure 19. The tumor-bearing mice showed good tolerance to the test drug 2E5 at all doses, and there was no significant weight loss in all treatment groups. To sum up the above, in this experiment, the 3mg/k group and the 10mg/kg group of the test substance 2E5 had significant anti-tumor effects on the CloudmanS91 subcutaneous syngeneic tumor model, but there was no dose-dependence, and the 3mg/kg dose group had anti-tumor effects. Tumor effect is better than 10mg/kg dose group.

The above has been described based on the embodiments of the present invention, but the present invention is not limited thereto, and those skilled in the art should understand that modifications and changes can be made within the scope of the gist of the present invention. The way of modification should belong to the protection scope of the present invention.

Claims (34)

1. An antibody or an antigen-binding fragment thereof, which binds to an epitope of PD-1, said epitope comprising: amino acids at positions 128, 129, 130, 131 and 132 on SEQ ID NO: 24 and at position 35 , at least one amino acid in positions 64, 82, and 83. 2. An antibody or an antigen-binding fragment thereof, which binds to an epitope of human PD-1 and mouse PD-1, wherein the epitope comprises SEQ ID NO: 24 on the 128th, 129th, 130th, 131st and 132-position amino acid. 3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the murine PD-1 is mouse or rat PD-1. 4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein said antibody a) bind to human _PD -1 with a KD of 2.15E-10M or less; and b) Binding to mouse PD-1, K _D is below 1.67E-08M. 5. The antibody of any one of claims 1 to 4, wherein the antibody has at least one of the following properties: a) Binding to human PD-1 with a K _D of 4.32E-10M to 2.15E-10M, and binding to mouse PD-1 with a K _D of 5.39E-08M to 1.67E-08M; b) substantially not binding to human CD28, CTLA-4; c) increasing the proliferation of T cells; d) increase the production of interferon-gamma; or e) Increased secretion of interleukin-2. 6. An antibody or antigen-binding fragment thereof comprising an amino acid sequence that is the same as that selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9 the sequences have at least 70%, 80%, 90% or 95% homology, Wherein the antibody specifically binds to PD-1. 7. An antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9 , Wherein the antibody specifically binds to PD-1. 8. An antibody or antigen-binding fragment thereof comprising: a) a heavy chain variable region having an amino acid sequence at least 70%, 80%, 90% or 95% homologous to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 sex; and b) a light chain variable region having an amino acid sequence at least 70%, 80%, 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8 and 9 or 95% homology, Wherein the antibody specifically binds to PD-1. 9. An antibody or antigen-binding fragment thereof comprising: a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; and b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8 and 9, Wherein the antibody specifically binds to PD-1. 10. An antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-23, Wherein the antibody specifically binds to PD-1. 11. An antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences; and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, Wherein the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13 and conservative modifications thereof, Wherein the antibody specifically binds to PD-1. 12. The antibody of claim 11, wherein the antibody light chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22 and 23 and conservative modifications thereof. 13. The antibody of claim 11 or 12, wherein the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and conservative modifications thereof. 14. The antibody according to any one of claims 11 to 13, wherein the light chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19 and conservative modifications thereof. 15. The antibody according to any one of claims 11 to 14, wherein the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and conservative modifications thereof. 16. The antibody of any one of claims 11 to 15, wherein the light chain variable region CDR1 sequence comprises amino acids selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17 and 18 Sequences and their conservative modifications. 17. The antibody of any one of claims 1 to 16, wherein the antibody is a chimeric or humanized or human antibody. 18. The antibody of any one of claims 6 to 17, wherein said antibody exhibits at least one of the following properties: a) the KD for binding to human _PD -1 is 2.15E-10M or less, and the KD for binding to mouse _PD -1 is 1.67E-8M or less; b) substantially not binding to human CD28, CTLA-4; c) increase T cell proliferation; d) increase the production of interferon-gamma; or e) Increased secretion of interleukin-2. 19. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-18. 20. A cloning or expression vector comprising the nucleic acid molecule of claim 19. 21. A host cell comprising more than one cloning or expression vector as claimed in claim 20. 22. A process for producing the antibody of any one of claims 1 to 18, comprising culturing the host cell of claim 21, and isolating the antibody. 23. The process of claim 22, wherein the antibody is prepared by immunizing SD rats with the extracellular domain of human PD-1 and the extracellular domain of mouse PD-1. 24. A transgenic rat comprising human immunoglobulin heavy chain and light chain transgenes, wherein the rat expresses the antibody of any one of claims 1-18. 25. A hybridoma prepared from the rat of claim 24, wherein said hybridoma produces said antibody. 26. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, and one or more pharmaceutically acceptable excipients, diluents or carriers. 27. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-18 linked to a therapeutic agent. 28. A pharmaceutical composition comprising the immunoconjugate of claim 27 and a pharmaceutically acceptable excipient, diluent or carrier. 29. A method for preparing an anti-PD-1 antibody or antigen-binding fragment thereof, comprising: (a) provide: (i) an antibody sequence of a heavy chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NO: 10, a CDR2 sequence selected from the group consisting of SEQ ID NO: 11 and a CDR2 sequence selected from the group consisting of SEQ ID NO: 11 ID NO: CDR3 sequences in the group consisting of 12 and 13; and/or (ii) an antibody sequence of a light chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17 and 18, selected from the group consisting of SEQ ID: 19 and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22 and 23; and (b) Expression changes the antibody sequence into a protein. 30. A method of modulating an immune response in a subject comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of claims 1-18. 31. Use of the antibody according to any one of claims 1 to 18 for the preparation of a medicament for the treatment or prevention of immune disorders or cancer. 32. A method of inhibiting tumor cell growth in a subject, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-18 to the subject to inhibit tumor cell growth . 33. The method of claim 32, wherein the tumor cells are selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer Cancer in the group consisting of malignant melanoma of the skin or eye, uterus, ovary, and rectum. 34. The method of claim 32 or 33, wherein the antibody is a chimeric antibody or a humanized antibody.