CN111704671B

CN111704671B - OX40 antibodies and their use in treating cancer

Info

Publication number: CN111704671B
Application number: CN202010834165.4A
Authority: CN
Inventors: 史辛艺; 王泰华; 刘欢; 戴伟利
Original assignee: Guangdong Cel Biotechnology Co Ltd
Current assignee: SHANDONG NEW MEDICINE RESEARCH INSTITUTE OF INTEGRATED TRADITIONAL AND WESTERN MEDICINE LLC
Priority date: 2020-08-19
Filing date: 2020-08-19
Publication date: 2020-11-24
Anticipated expiration: 2040-08-19
Also published as: CN111704671A

Abstract

The invention relates to an OX40 antibody and application thereof in treating cancer, the antibody can better combine with human OX40, simultaneously promote T cell activation and subsequent IL-2 secretion and T cell proliferation, induce T cell mediated antitumor activity, and has wide application prospect.

Description

OX40 antibodies and their use in treating cancer

Technical Field

The invention relates to the field of biomedicine, in particular to an OX40 antibody and application thereof in treating cancer.

Background

OX4O (CD134) is a type I transmembrane glycoprotein with a molecular weight of 48-50kD, a member of the TNFR superfamily, expressed predominantly in activated CD4⁺T cell surface, at CD8⁺There is also a small amount of expression on the surface of T cells, activated regulatory T cells, natural killer T (nkt) cells, NK cells and neutrophils. The ligand OX4OL (CD134L) is a member of TNF superfamily, a type II transmembrane glycoprotein with a molecular weight of 34-40kD, and is mainly expressed in mature Dendritic Cells (DC), activated B cells, Vascular Endothelial Cells (VEC), umbilical vein vascular endothelial cells (HUVEC), macrophages and certain tissues and organs including heart, skeletal muscle, innocent pill, lung and the like. Co-stimulatory signals mediated by OX40/0X40L are involved in T cell activation, proliferation and migration, maintenance of long-term T cell production, and promotion of germinal center formation in conjunction with CD28 signaling. CD28/B7 plays a major role in primary immune responses, while OX40/OX4OL signals play important regulatory effects in the late stages of primary immunity, secondary immune responses and immunological memory.

The presence of OX40+ T cells was found by Vetto et al in melanoma, infiltrating lymphocytes TILs (31%) of head and neck phosphorus cancer and tumor ductal lymph node cells DLNCs (28%). Animal experiments prove that the exciting OX40 monoclonal antibody can prevent tumor formation of mice and improve the survival rate of tumor-bearing mice. The combined use of OX40 monoclonal antibody and IL-2 also has good effect in adoptive immunotherapy of mouse tumor models, so that the metastasis in lung and brain is eliminated. In a plurality of tumor models of mice, including sarcoma, breast cancer, intestinal cancer, glioma, melanoma and the like, OX40/OX40L immune intervention strategies are successfully applied. As OX40 is mainly expressed on antigen-specific T cells at inflammatory sites, the OX40 becomes an ideal target molecule for immune intervention, and the anti-tumor capability of a body is expected to be improved by activating OX4O signals in vivo, promoting the secretion of cytokines and increasing the number of antigen-specific memory cells. The development of OX40 antibodies is of great clinical importance, and there are some published OX40 antibodies in the prior art, such as US20160137740A, WO2015153513A, CN110078825B, CN111196854A, CN108623686, etc., but there is still a great need for new anti-OX 40 antibodies.

Disclosure of Invention

Based on the above findings, the present invention provides an agonistic OX40 antibody that is able to better bind human OX40 while promoting T cell activation and subsequent IL-2 secretion and T cell proliferation, inducing T cell-mediated anti-tumor activity.

The invention provides the following technical scheme:

a humanized OX40 antibody comprising a heavy chain variable region and a light chain variable region, wherein the light and heavy chain variable region each has 3 Complementarity Determining Regions (CDRs). The 3 CDR sequences of the heavy chain variable region of the OX40 antibody are respectively SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; the variable region of light chain has 3 CDR sequences of SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7.

In some embodiments, the OX40 antibodies of the invention comprise a heavy chain variable region sequence as SEQ ID NO. 4 and a light chain variable region sequence as SEQ ID NO. 8.

In some embodiments, the OX40 antibodies of the invention comprise a heavy chain comprising a heavy chain variable region and a heavy chain constant region and a light chain comprising a light chain variable region and a light chain constant region.

In some embodiments, the OX40 antibody light chain constant region of the invention comprises a human kappa and lambda chain sequence, and more preferably a kappa chain. The antibody heavy chain constant region comprises human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM, and more preferably IgG 1.

In some embodiments, the OX40 antibodies of the invention have agonist activity. Agonist activity of anti-OX 40 antibodies was assessed by the level of cytokine released after T cell activation. The cytokine is an inflammatory cytokine, for example assessed by levels of interferon-gamma (e.g., IFNg), interleukin 2 (e.g., IL-2), interleukin 10 (e.g., IL-10).

In some embodiments, the OX40 antibodies of the invention have agonist activity. Agonist activity of anti-OX 40 antibodies was assessed by T cell proliferation levels.

In some embodiments, binding of an OX40 antibody described herein to human OX40 is determined by an Elisa or flow cytometry (e.g., FACS) assay.

The present invention provides a pharmaceutical combination comprising an OX40 antibody of the invention, in addition to a second drug or active agent. The second agent may be a CTLA4 antibody, a PD-1 antibody, a PD-L1 antibody, a 4-1BB antibody, or other immunotherapeutic agent.

The OX40 antibodies of the invention are useful in the preparation of a medicament for the treatment and/or prevention of a cancer selected from the group consisting of: breast cancer, melanoma, B cell lymphoma, head and neck cancer, colon cancer, prostate cancer, lung cancer.

Advantageous effects

The invention has the following beneficial effects: provided are novel agonistic OX40 antibodies that are capable of better binding to human OX40 while promoting T cell activation and subsequent IL-2 secretion and T cell proliferation, inducing T cell-mediated antitumor activity.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 Proliferative effects of anti-OX 40 antibodies on T cells.

FIG. 2 Effect of anti-OX 40 antibodies on cytokine secretion by T cells.

FIG. 3 anti-tumor effect of anti-OX 40 antibody.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Example 1 anti-OX 40 antibody preparation and purification

Recombinant human OX40 extracellular domain (OX40-ECD) protein was used as immunogen (purchased from Cat #: 10481-H08H, Chinesia). Dissolving the immune antigen in PBS, and uniformly mixing with Freund's adjuvant with the same volume to form the water-in-oil emulsion. The primary immunization was performed by subcutaneous multiple immunization at a dose of 100. mu.g/mouse. 7-10 days after the initial immunization, mice were immunized in the same way and at the same dose by mixing and emulsifying incomplete Freund's adjuvant and immunizing antigen in equal volume. Then boosting the immunity for several times every 7-10 days to make the antibody titer of the mouse serum reach more than ten thousand. One week after the last booster immunization, an eye bleed was performed and the serum titer was determined by ELISA. If the serum titer of the mice does not reach ten thousand, the boosting is required to be continuously performed for several times. And (3) carrying out impact immunization on mice with the titer reaching more than ten thousand three days before fusion, taking antigen without adjuvant, and injecting the antigen into tail vein.

The mouse myeloma cells SP2/0 were recovered 10-14d before fusion and cultured in DMEM medium containing 10% FCS. The cell concentration was adjusted to 3X105/m1 with fresh medium 24-36h before fusion. DMEM and PEG solution were pre-warmed in a 37 deg.C water bath prior to fusion. 2x107 well-grown SP 20/cells were collected, washed with DMEM 2 times, mixed well with (1-2) x108 spleen cells in a 50ml transparent plastic centrifuge tube, washed again with DMEM, centrifuged at 1OO0rPm for 5min, the supernatant was discarded, and the cells were gently loosened to give a paste. Placing the centrifuge tube in a 37 ℃ thermos cup, preheating for 5min, sucking 50% PGE solution of lml subjected to 37 ℃ preheating, gently inserting a suction tube into the bottom of the cells, adding the fusion agent in the lmin at a constant speed while stirring, and then standing the lmin. Then adding DMME preheated at 4Om 137 ℃, slowly and quickly stirring while adding, and then standing for 5-10min at 37 ℃. After centrifugation at 800rpm for 10min, the supernatant was discarded and the cells were resuspended in 55-60 ml of 37 ℃ pre-warmed DMEM medium containing 1% HAT, 20% FCS. After gently mixing, the mixture was dropped into a 96-well plate containing feeder cells, 60. mu.l of the mixture was cultured at 37 ℃ in 5% C02, and the culture solution was changed after 4 to 5 days. And when the cell clones are full of l/3 culture holes, sucking culture supernatant, and screening positive hybridoma cell strains by adopting indirect ELISA. Screening monoclonal positive hybridoma cell strain by limiting dilution method, enlarging culture of positive hybridoma cell, and freezing for seed preservation. The finally screened positive hybridoma cell line was named 4F 9-2.

Example 2 antibody humanization

The hybridoma cell line 4F9-2 obtained in example 1 was lysed with TRNzol-A +, and the cell line was placed in a centrifuge tube, 200. mu.l of chloroform was added to each ml of TRNzol-A +, and the tube was vortexed for 15 seconds and left for 3 minutes. 13000 rpm, 4 ℃ for 10 minutes, Trizol-A + cell solution is divided into three layers: transferring the water phase dissolved with the RNA into a centrifuge tube, adding isopropanol with the same volume into the water phase, uniformly mixing, and standing at room temperature for 25 minutes. 13000 rpm, 4 ℃ for 10 minutes, discard the waste liquid to get the bottom RNA precipitation. After washing the RNA pellet twice with 75% ethanol, the RNA was dissolved in DEPC water and stored at-80 ℃. Then, using a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TRANS, AT311-02) reverse transcription kit to synthesize a first strand of cDNA; using cDNA as template, amplifying DNA product containing antibody heavy chain variable region and light chain variable region by PCR, separating and recovering purified target fragment containing antibody heavy chain variable region and light chain variable region by agarose gel electrophoresis. Cloning the DNA into pGEM-T vector, screening positive clone sequencing, and further obtaining the amino acid sequence corresponding to the variable region gene according to the sequencing result.

According to the variable region sequence of the antibody secreted by hybridoma cell strain 4F9-2, the antibody is humanized and reformed, firstly, the VH region and VK region of hybridoma cell strain 4F9-2 are compared with the sequences of the existing humanized antibody in NCBI database by utilizing the currently effective public database, and then the sequences of the existing humanized antibody in the NCBI database are selected

The closest human embryonic framework sequences to which the CDR regions are grafted onto the selected human antibody framework coding sequences to produce the humanized antibody. Then determining some key amino acids for maintaining the binding affinity and the space frame, grafting the amino acids back to the selected human embryonic line antibody framework, and determining the light-heavy chain variable region sequence of the finally modified humanized antibody. Antibody VH and VK domains were synthesized and cloned into a specialized mammalian expression vector that enabled expression of the respective domains in the complete human IgG1 or kappa antibody backbone. The lgG 1 construct was co-transfected with the kappa construct into 293F cells for small-scale preparation of humanized antibodies. The supernatant of the transient transfection was passed through a protein A or G resin to obtain purified OX40 antibody. Finally, the humanized and transformed antibody is named as h4F9-2, and the sequencing result shows that the amino acid sequences of VH-CDR1-3 of the antibody h4F9-2 are respectively shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3; the amino acid sequence of the antibody VH is shown in SEQ ID NO. 4; the amino acid sequences of VL-CDR1-3 of h4F9-2 are respectively shown as SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7; the amino acid sequence of the antibody VL is shown in SEQ ID NO. 8.

Example 3 affinity assay for humanized antibodies

After equilibration of the chip with HBS-EP buffer, the carboxymethylated dextran biosensor chip, i.e. the CM5 chip, was activated with N-ethyl-N' - (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Antigen protein (OX 40-HIS) was diluted to 5. mu.g/ml with 10mM sodium acetate pH4.8, followed by 3min injection at a flow rate of 10. mu.l/min for antigen coupling, and ethanolamine at a flow rate of 10. mu.l/min for blocking unreacted groups. The anti-OX 40 antibody hF9-2 and the control antibody tavolimab were subjected to 6 different concentration gradients, and sequentially subjected to 2-fold gradient dilution with HBS-EP buffer at a concentration of 100nM to 1.56 nM. Sequence of control antibody tavolimab reference IMGT/mAb-DB ID: 608 (see: www.imgt.org/mAb-DB/search. action). Starting from low concentrations, anti-OX 40 antibody was injected at a flow rate of 50. mu.L/min for 3min, followed by HBS-EP buffer with a dissociation time of 10min at a flow rate of 50. mu.L/min. Use of 3M MgCl₂As a regeneration buffer, the chip was regenerated according to the regeneration procedure. Thereafter, other concentrations of the antibody were measured in the same manner. Calculation of binding Rate (K) by Simultaneous fitting of binding and dissociation sensorgrams_a) And dissociation Rate (K)_d). Equilibrium dissociation constant (K)_d) Using dissociation rate (K)_d) Rate of binding (K)_a) And (4) calculating.

Watch 1

Antibodies	K_a（1/Ms）	K_d（1/s）	K_D(M)
				h4F9-2	3.66×10⁵	2.57×10^-4	7.02×10^-10
tavolimab	3.31×10⁵	8.49×10^-4	2.56×10^-9

Example 4 proliferation-promoting Effect of OX40 antibody on T cells

Collecting fresh peripheral blood of normal human anticoagulated with heparin sodium, and performing conventional Ficoll (purchased from GE Healthcare) gradient centrifugation to obtain mononuclear cells (PBMC) with cell concentration adjusted to 2x10⁶M1, adding 10% SRBC-AET cell suspension (volume ratio of 2:1), mixing, placing in 37 deg.C water bath for 15min, centrifuging at low speed (500rpm) for 5min, transferring to 4 deg.C refrigerator for 45min, diluting with PBS, centrifuging with Ficoll density gradient, precipitatingThe cells were treated with ACK solution (NH per 100ml solution)₄Cl 0.83g,NaHCO₃0.1g, EDTA-Na 3.7g) to lyse erythrocytes, and generally repeating the lysis 2 to 3 times, to finally obtain PBTC cells. anti-CD 3 antibody (purchased from Biolegend) was coated onto 96-well plates (1. mu.g/ml) at 50. mu.l per well overnight at 4 ℃. The supernatant was aspirated and washed 1 time with PBS. PBTC was adjusted to 0.8X10 in RMPI1640 medium with 10% FCS⁶The anti-CD 28 antibody (purchased from Biolegend) was adjusted to a final concentration of 2. mu.g/ml, inoculated into the 96-well plate (100. mu.l/well), and cultured at 37 ℃ for 24 hours in 5% C02. Then, 3 groups of 3 wells were added with 100. mu.l/well of anti-OX 40 antibody h4F9-2 (final concentrations 0.5, 1, 2. mu.g/m 1, respectively) at different concentrations, and controls were set in which tavolimab was used as a positive control and IgG1 was used as a negative control antibody. After further culturing for 72h, 20. mu.l MTT (5mg/ml) solution was added to each well, culturing was further carried out for 4-6h, the supernatant was aspirated off, washed once with 200. mu.l/well PBS, dissolved by adding 100 ul/well isopropanol, left to stand for 5min, and the 0D was measured₅₇₀The value is obtained. The experimental results are shown in fig. 1: the anti-OX 40 antibody h4F9-2 and the tavolimab can promote the cell proliferation of T cells in a dose-dependent manner, and the anti-OX 40 antibody h4F9-2 has better effect on promoting the cell proliferation of the T cells.

Example 5 Effect of OX40 antibodies on cytokine secretion by T cells

Taking fresh normal human peripheral blood anticoagulated by heparin sodium, obtaining mononuclear cells (PBMC) according to a conventional Ficoll (purchased from GE Healthcare) gradient centrifugation method, adjusting the cell concentration to 2x106/m1, adding 10% SRBC-AET cell suspension (the volume ratio is 2:1), uniformly mixing, placing in a 37 ℃ water bath for 15min, then carrying out low-speed centrifugation (500rpm) for 5min, transferring to a4 ℃ refrigerator for combination for 45min, diluting by PBS, carrying out Ficoll density gradient centrifugation, dissolving erythrocytes by an ACK solution (each 100ml of the solution contains NH4Cl 0.83.83 g, NaHCO 30.1g and EDTA-Na 3.7g), generally repeatedly dissolving for 2-3 times, and finally obtaining the PBTC cells. anti-CD 3 antibody (purchased from Biolegend) was coated onto 96-well plates (1. mu.g/ml) at 50. mu.l per well overnight at 4 ℃. The supernatant was aspirated and washed 1 time with PBS. PBTC was adjusted to 0.8X10 in RMPI1640 medium with 10% FCS⁶The anti-CD 28 antibody (purchased from Biolegend) was adjusted to a final concentration of 2. mu.g/ml, inoculated into the 96-well plate (100. mu.l/well), and cultured at 37 ℃ for 24 hours in 5% C02. Then, 100. mu.l/well of anti-OX 40 antibody h4F9-2 (final concentrations 0.4, respectively),2. 10 ug/m 1), 3 wells in each group, with controls, in which tavolimab was used as a positive control and IgG1 was used as a negative control antibody. After further culturing for 72 hours, culture supernatants were collected and the secretion of the cytokine IL2 was measured. The cytokine content was determined according to the procedure described for the ELISA kit. The experimental results are shown in fig. 2: the anti-OX 40 antibodies h4F9-2 and tavolimab can improve the secretion of IL-2 in T cells in a dose-dependent manner and enhance the activity of the T cells.

Example 6 in vivo efficacy of anti-OX 40 antibodies

To investigate the in vivo anti-tumor effect of OX40 monoclonal antibody h4F9-2, a MC38 colon cancer model was constructed from 6-8 week-sized female OX40 humanized mice (purchased from Beijing Baiosaccae Gene Biotechnology, Inc.). Sufficient MC38 tumor cells in log phase were cultured in DMEM medium, the cells were collected and washed with PBS, followed by dilution to 1x10 cell concentration⁷One per ml. 100. mu.l of MC38 cell suspension was inoculated subcutaneously into the abdomen of mice. When the MC38 tumor grows to about 80m in volume³At the time, we randomly divided tumor-bearing nude mice into 3 groups of 6 mice each and administered the experiments, including: (a) group 1: PBS (vehicle control); (b) group 2: anti-OX 40 antibody h4F9-2, 5 mg/kg; (c) group 3: anti-OX 40 control antibody h4F9-2, 5 mg/kg; the routes of administration are: anti-OX 40 antibody was administered 1 time every 3 days, 7 times weekly by intravenous injection at the trailing edge. Measuring the length and the short diameter of the tumor every 3 days, and calculating the size of the tumor by the following formula: volume of transplanted tumor = major diameter of tumor x broad diameter of tumor x 0.5. When the whole experiment reaches the observation end point, the nude mice are sacrificed by cervical dislocation. The experimental results are shown in fig. 3, and the tumor volume of the nude mice of the negative control group is mostly in progressive growth. The anti-OX 40 antibody h4F9-2 and the positive control antibody tavolimab both significantly inhibited tumor growth.

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Claims

1. An anti-OX 40 antibody, which consists of a heavy chain and a light chain, wherein 3 CDR sequences in a heavy chain variable region of the antibody are respectively SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3; the variable region of light chain has 3 CDR sequences of SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7.

2. The anti-OX 40 antibody of claim 1, characterized by: the heavy chain comprises a heavy chain variable region as shown in SEQ ID No. 4; the light chain comprises a light chain variable region as shown in SEQ ID No. 8.

3. A pharmaceutical composition characterized by: comprising the antibody of any one of claims 1-2.

4. Use of an anti-OX 40 antibody of any one of claims 1-2 in the manufacture of a medicament for treating colon cancer.