CN115850471A

CN115850471A - Anti-human IL-2 monoclonal antibody and application thereof

Info

Publication number: CN115850471A
Application number: CN202211255719.0A
Authority: CN
Inventors: 娄竞; 陈建鹤; 苏冬梅; 靳征; 吕云英; 张若兰; 裴若辰; 欧艳梅; 曲啸; 谢写; 张静; 曾淋
Original assignee: Shenzhen Bestone Technology Development Co ltd
Current assignee: Shenzhen Bestone Technology Development Co ltd
Priority date: 2022-10-13
Filing date: 2022-10-13
Publication date: 2023-03-28
Also published as: WO2024077773A1

Abstract

The invention belongs to the technical field of antibody engineering, relates to an anti-human IL-2 monoclonal antibody and application thereof, and particularly relates to an anti-human IL-2 monoclonal antibody, and preparation and application of an antibody compound. After the anti-human IL-2 monoclonal antibody provided by the invention is combined with IL2, the combination of IL2 and IL2R beta/gamma (CD 122/132) is kept, meanwhile, the combination of IL-2 and IL-2R alpha (CD 25) can be blocked, and the growth of transplanted tumors in mice can be effectively inhibited. Meanwhile, the invention introduces mutation into Fc of anti-human IL-2 monoclonal antibody, and after the Fc mutated monoclonal antibody and IL-2 form a compound, the in vivo anti-tumor activity of the IL-2/antibody compound can be retained, side effects can be greatly reduced, and the safety of the medicine can be improved.

Description

Anti-human IL-2 monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of antibody engineering, relates to an anti-human IL-2 monoclonal antibody and application thereof, and particularly relates to an anti-human IL-2 monoclonal antibody, preparation of an antibody compound and application thereof.

Background

Interleukin-2 (IL-2) is a cytokine with strong immunological activity, has effects on T cell activation and growth, and participates in antitumor effect and graft rejection. The medicines aiming at IL-2 are more, such as aldesleukin and the like, and have a long history when being clinically used for treating malignant tumors. However, in the clinical application of IL-2, more toxic and side effects are found, particularly the toxicity to the liver and the lung, which limits the application. Moreover, it was found during the application of IL-2 that it can stimulate Treg proliferation and cause immune cells to undergo activation-induced death (AICD), further affecting its therapeutic efficacy.

IL-2 acts by binding to the IL-2 receptor, which consists of three subunits, α, β, and γ, where α is a high affinity receptor and β and γ, although low affinity receptors, mediate signaling upon binding of IL-2 to the receptor. The antitumor activity of IL-2 is mainly achieved by activating CD8 positive T cells and NK cells, but it simultaneously expands CD4 positive Treg cells, reducing or even completely eliminating its antitumor effect.

The CD4 positive Treg cells highly express IL-2R alpha (CD 25), and because CD25 is a high affinity receptor of IL-2, the IL-2 at low concentration can preferentially act and bind to the Treg cells, and can combine with the CD25 to play an anti-tumor immunosuppressive role. CD8 positive T cells and NK cells are affected by IL-2 at high concentrations because they do not express or express low alpha receptors, but express high beta and gamma receptors. Therefore, it is an effective means to achieve IL-2 antitumor effect by reducing the binding of IL-2 to α receptor or enhancing the binding of IL-2 to β/γ receptor to bias IL-2 toward CD 8-positive T cells and NK cells.

However, it has been found that whether IL-2 monoclonal antibody, IL-2 and Fc fusion protein or antibody-IL-2 bifunctional molecule is used for treating malignant tumor, there are some toxic and side effects, including liver toxicity. Therefore, the research and development of an IL-2 medicament with low toxic and side effects and high antitumor activity is a problem to be solved urgently at present.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides an anti-human IL-2 monoclonal antibody, preparation of an antibody compound and application thereof. The invention takes recombinant human IL-2 protein as immunogen, prepares mouse anti-human IL-2 monoclonal antibody m22F8 by hybridoma technology, analyzes the binding activity of the mouse antibody, determines the affinity of IL-2, analyzes the effect of the mouse antibody for blocking IL-2 from binding CD25, and prepares the anti-human IL-2 chimeric antibody by determining the gene of the mouse hybridoma antibody. On the basis of performance analysis of a mouse antibody and a chimeric antibody, a CDRs transplantation technology and CDR region mutation design are adopted to construct a humanized anti-human IL-2 monoclonal antibody, the affinity of the humanized antibody to IL-2 is detected, the effect of blocking IL-2 from combining with CD25 is analyzed, the anti-tumor activity of an anti-human IL-2 monoclonal antibody m22F8 and an antibody compound and the research on the synergic anti-tumor activity of the antibody compound and a PD1 monoclonal antibody have good application prospect to anti-tumor medicaments.

The present invention provides an anti-human IL-2 monoclonal antibody having:

a heavy chain variable region having an amino acid sequence of SEQ ID NO. 4, and a light chain variable region having an amino acid sequence of SEQ ID NO. 9.

Further, the nucleotide encoding the anti-human IL-2 monoclonal antibody has:

the nucleotide sequence of the heavy chain variable region as shown in SEQ ID NO. 3; the nucleotide sequence of the light chain variable region shown as SEQ ID NO. 8.

Further, the antibody is derived from a murine antibody.

The present invention provides an anti-human IL-2 chimeric antibody having:

a heavy chain sequence having the amino acid sequence of SEQ ID NO. 15, and a light chain sequence having the amino acid sequence of SEQ ID NO. 16.

The present invention also provides an Fc mutant anti-human IL-2 chimeric antibody having: a heavy chain sequence having the amino acid sequence of SEQ ID NO. 18, and a light chain sequence having the amino acid sequence of SEQ ID NO. 16.

The preparation method comprises the following steps: converting the 310 th site H of a human IgG4 constant region into A and converting the 435 th site amino acid H into Q by adopting a gene site-directed mutagenesis technology or a gene synthesis technology to form a mutated human IgG4 constant region, wherein the amino acid sequence is shown as SEQ ID NO. 17; the heavy chain variable region of the anti-human IL-2 monoclonal antibody is recombined with a mutated human IgG4 constant region to form the heavy chain of the Fc mutated anti-human IL-2 chimeric antibody, and the amino acid sequence is shown as SEQ ID NO 18; recombining the variable region sequence of the light chain of the antihuman IL-2 monoclonal antibody with the human kappa chain constant region with the amino acid sequence shown as SEQ ID NO. 14 to form the light chain of the antihuman IL-2 chimeric antibody, wherein the amino acid sequence is shown as SEQ ID NO. 16; then constructed into pcDNA3.4 expression vector, transfected Expi-293F cell and purified by Protein G to obtain Fc mutant anti-human IL-2 chimeric antibody.

Furthermore, the invention also provides a humanized anti-human IL-2 monoclonal antibody, which is constructed by adopting CDRs transplantation technology and CDR region mutation design on the basis of the anti-human IL-2 monoclonal antibody.

Further, the humanized anti-human IL-2 monoclonal antibody has: the heavy chain variable region having the amino acid sequence of SEQ ID NO. 24 and the light chain variable region having the amino acid sequence of SEQ ID NO. 25.

The present invention also provides a humanized anti-human IL-2 antibody having:

a heavy chain sequence having the amino acid sequence of SEQ ID NO. 27, and a light chain sequence having the amino acid sequence of SEQ ID NO. 28.

The present invention also provides an Fc mutant humanized anti-human IL-2 antibody having:

the heavy chain sequence having the amino acid sequence of SEQ ID NO. 26 and the light chain sequence having the amino acid sequence of SEQ ID NO. 28.

The preparation method comprises the following steps: recombining the heavy chain variable region of the humanized anti-human IL-2 monoclonal antibody with a mutant IgG4 constant region with the amino acid sequence shown as SEQ ID NO:17 to obtain the heavy chain of the humanized anti-human IL-2 antibody with Fc mutation, wherein the amino acid sequence is shown as SEQ ID NO: 26; the light chain variable region sequence of the antihuman IL-2 monoclonal antibody is recombined with the human kappa chain constant region with the amino acid sequence shown as SEQ ID NO. 14 to obtain the light chain of the humanized antihuman IL-2 chimeric antibody, and the amino acid sequence is shown as SEQ ID NO. 28; then constructed into pcDNA3.4 expression vector, transfected Expi-293F cell, and purified by Protein G to obtain Fc mutant humanized anti-human IL-2 antibody.

The invention also provides an antibody compound which is prepared by mixing IL-2 (interleukin-2) and the anti-human IL-2 chimeric antibody or Fc mutant anti-human IL2 chimeric antibody or humanized anti-human IL-2 antibody or Fc mutant humanized anti-human IL-2 antibody obtained by the invention according to the mass ratio of 1:7.

In addition, the invention also provides application of the antibody compound in preparing anti-tumor drugs.

Compared with the prior art, the anti-human IL-2 monoclonal antibody prepared by the invention keeps the combination with IL2R beta/gamma (CD 122/132), can block the combination of IL2 and IL2R alpha (CD 25), and can effectively inhibit the growth of transplanted tumors in mice. Meanwhile, the mutation is introduced into the Fc of the IL-2 monoclonal antibody, and after the Fc mutated monoclonal antibody and the IL-2 form a compound, the in-vivo anti-tumor activity of the IL-2/antibody compound can be retained, the side effect can be greatly reduced, and the safety of the medicine is improved.

For a better understanding of the present invention, certain terms are first defined. Other definitions are listed throughout the detailed description section.

The term "IL-2" is interleukin-2, a cytokine of the chemokine family, IL-2 has a molecular weight of 15kD, is a glycoprotein containing 113 amino acid residues, and is encoded by a gene on chromosome 4 in humans.

The term "antibody" herein is intended to include full-length antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof. Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated VL) and a light chain constant region (abbreviated CL).

The term "monoclonal antibody" or "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "EC50", also known as half maximal effect concentration, refers to a concentration that causes 50% of the maximal effect.

The term "IC50", also known as the semi-inhibitory concentration, refers to the concentration of a drug or inhibitor required to inhibit a given biological process or a component of the biological process (e.g., an enzyme, receptor, cell, etc.) by half.

Description of the drawings:

FIG. 1 is a diagram showing the affinity of murine antibody m22F8 for the target antigen IL-2;

FIG. 2 is a graph of murine antibody m22F8 blocking human IL-2 binding to CD 25-ECD;

FIG. 3 is a graph of the anti-tumor activity of the IL-2/m22F8 complex;

FIG. 4 is a graph of serum ALT assay of surviving mice dosed 2 times with IL-2/ch22F8mu complex at 3mg/kg based on IL-2;

FIG. 5 is a graph of serum ALT assay of surviving mice dosed 6 times with 1mg/kg dose of IL-2/ch22F8mu complex as IL-2;

FIG. 6 is a graph of the in vivo anti-tumor activity of the IL-2/ch22F8mu complex;

FIG. 7 is a graph of ch22F8mu and hu22F8mu binding to human IL-2;

FIG. 8 is a graph of hu22F8mu and ch22F8mu blocking IL-2 binding to CD 25;

FIG. 9 is a graph of IL-2/hu22F8mu complex stimulation of CTLL2 cell proliferation;

FIG. 10 is a graph of the effect of IL-2/hu22F8mu complex and IL-2/hu22F8 on binding to human FcRn/β 2M;

FIG. 11 is a graph of the effect of IL-2/hu22F8mu complex and IL-2/hu22F8 on binding to murine FcRn/β 2M;

FIG. 12 is a graph of the in vivo antitumor activity of the IL-2/hu22F8mu complex;

FIG. 13 is a graph of the anti-tumor effect of SPGD01 (IL-2/hu 22F8 mu);

FIG. 14 is a graph of the effect of SPGD01 (IL-2/hu 22F8 mu) on serum ALT in experimental mice;

FIG. 15 is a graph showing the effect of SPGD01 (IL-2/hu 22F8 mu) on the composition of CD4+/CD8+ lymphocytes in experimental animals;

FIG. 16 is a graph showing the effect of SPGD01 (IL-2/hu 22F8 mu) on the composition of CD4+/CD25+ lymphocytes in experimental animals;

FIG. 17 is a graph of the stability of SPGD01 (IL-2/hu 22F8 mu) and hu22F8 mu;

FIG. 18 is a graph of the stability of SPGD01 (IL-2/hu 22F8 mu) and hu22F8mu

FIG. 19 is a graph of the stability of SPGD01 (IL-2/hu 22F8 mu) and hu22F8mu

FIG. 20 is a graph of the stability of SPGD01 (IL-2/hu 22F8 mu) and hu22F8 mu.

Detailed Description

The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.

Example 1 preparation and screening of antigen-immunized mice and hybridomas

The method comprises the following steps: human IL-2 protein (purchased from Sino biological Co., ltd., good No. GMP-11848-HNAE, amino acid sequence shown as SEQ ID NO: 1) expressed by prokaryotic cells Escherichia coli was used for conventional immunization of Balb/c mice (purchased from Shanghai Ling Chang Biotech Co., ltd.); on day 1, emulsifying IL-2 protein with Freund complete adjuvant, performing subcutaneous multi-point injection (human IL-2 protein, 50 mu g/mouse/0.5 ml) on Balb/c mice, on day 21, emulsifying human IL-2 protein with Freund incomplete adjuvant, performing subcutaneous injection (human IL-2 protein, 50 mu g/mouse/0.5 ml) on Balb/c mice, on day 41, performing intraperitoneal injection excitation on human IL-2 protein, 50 mu g/mouse/0.2 ml, and after 3-4 days, taking mice spleen for fusion experiment;

step two: 3-4 days after the last immunization of mice, the mouse spleen cells and mouse myeloma cells SP2/0 were electrofused by an electrofusion apparatus (from BTX), and the fused cells were mixed in a complete medium (i.e., RPMI1640 and DMEM F12 medium 1:1, mixed well, and then 1% Glutamine (Glutamine), 1% Sodium pyroltate (Sodium pyruvate), 1% MEM-NEAA (minimal essential medium-nonessential amino acid solution), 1% penicillin-streptomycin (penicillin-streptomycin), 50 μ M β -mercaptoethanol and 20 FBS (fetal bovine serum), all of which were purchased from Gibco, and suspended uniformly in 10% of 10 ⁵ Each cell/100. Mu.l/well was divided into 25 pieces of 96-well culture plates and cultured overnight, and the next day 100. Mu.l of complete medium containing 2 XHAT per well was added to each well, so that the culture medium in the 96-well plate was 200. Mu.l/well (containing 1 XHAT); after 7-12 days, supernatants were harvested and hybridoma wells positive for human IL-2 binding activity were screened by indirect enzyme-linked immunosorbent assay (ELISA) to obtain 442 positive wells in total. 25 positive wells were obtained by further screening by detecting the effect of hybridoma wells on blocking IL-2 binding to CD 25. Carrying out first and second rounds of subcloning on hybridoma holes which are positive for human IL-2 combination and block positive for IL-2 combination CD25 by a limiting dilution method to obtain a hybridoma cell strain named as SPGD01-22F8.

Wherein the indirect enzyme-linked immunosorbent assay method is used for screening the human IL-2 combinationThe method for positive hybridoma wells was as follows: the recombinant human IL-2 protein was diluted to 1. Mu.g/ml with coating solution (50 mM carbonate coating buffer, pH 9.6), 100. Mu.l/well added to the microplate, and coated overnight at 4 ℃. PBST washing plate 3 times, adding 200 u l/hole blocking solution (2% BSA-PBST), 37 degrees C placed 1h PBST washing plate 1 times for use. And sequentially adding the collected hybridoma supernatant into the closed enzyme label plate at 100 mu l/hole, and standing at 37 ℃ for 1h. PBST washing plate 3 times, adding HRP labeled goat anti-mouse IgG secondary antibody (purchased from Millipore, cat. AP 181P), standing at 37 deg.C for 30min; after PBST washing for 5 times, residual droplets were patted dry on absorbent paper, 100. Mu.l of TMB (from BD Co., cat. No. 555214) was added to each well, and the mixture was left for 5min at room temperature (20. + -. 5 ℃) in the dark; add 50. Mu.l 2M H per well ₂ SO ₄ And stopping the substrate reaction by the stop solution, reading the OD value at 450nm of the microplate reader, and analyzing the binding capacity of the antibody to be detected and the target antigen IL-2.

The method for detecting the hybridoma holes for blocking IL-2 from being combined with CD25 comprises the following steps: recombinant human CD25-ECD (purchased from Beijing Yiqiao Shenzhou Co., ltd., product No. 50292-M02H) was diluted to 1. Mu.g/ml with a coating solution (50 mM carbonate coating buffer, pH 9.6), 100. Mu.l/well was added to the microplate, and the mixture was coated overnight at 4 ℃. PBST washing plate 3 times, adding 200 u l/hole blocking solution (2% BSA-PBST), 37 degrees C placed 1h PBST washing plate 1 times for use. 60ul of each hybridoma supernatant collected was incubated with 80ul of biotin-labeled IL-2 (bio-IL-2, available from Sino biological Co., ltd., product No. 11848-HNAE-B) diluted to 10ng/ml at 37 ℃ for 30min, followed by addition of the blocked microplate, 100. Mu.l/well and standing at 37 ℃ for 1h. PBST washing plate 3 times, adding HRP-labeled SA (SA-HRP, pierce company), standing at 37 deg.C for 30min; after PBST plate washing 5 times, the residual liquid drop is patted dry on the absorbent paper, 100 μ l of TMB (purchased from BD company, cat # 555214) is added into each hole, and the plate is placed for 5min in the dark at room temperature (20 +/-5 ℃); add 50. Mu.l 2M H per well ₂ SO ₄ Stopping the substrate reaction by the stop solution, reading the OD value at 450nm of an enzyme-labeling instrument, and analyzing the effect of the to-be-detected antibody on blocking the IL-2 from being combined with CD 25.

Example 2 preparation of murine anti-human IL-2 monoclonal antibody m22F8

The hybridoma cell lines obtained were amplified and selected in complete medium (as described in example 1), centrifuged to remove the medium and cultured in serum-free mediumNutrient SFM medium (purchased from Life technologies, ltd., cat # 12045-076) to give a cell density of 1 to 2X 10 ⁷ Per ml at 5% CO ₂ Culturing at 37 deg.C for 1 week, centrifuging to obtain culture supernatant, and purifying by Protein G affinity chromatography to obtain murine anti-human IL-2 monoclonal antibody m22F8.

Example 3 measurement of affinity of murine anti-human IL-2 monoclonal antibody m22F8 for target antigen IL2

The affinity of the murine anti-human IL-2 monoclonal antibody m22F8 to the recombinant human IL-2 protein is determined by an ELISA method, and the experimental method is as follows:

the recombinant human IL-2 protein was diluted to 1. Mu.g/ml with coating solution (50 mM carbonate coating buffer, pH 9.6), 100. Mu.l/well added to the microplate, and coated overnight at 4 ℃. PBST washing plate 3 times, adding 200 u l/hole blocking solution (2% BSA-PBST), 37 degrees C placed 1h PBST washing plate 1 times for use. The murine anti-human IL-2 monoclonal antibody m22F8 was diluted to 5000/1000/200/40/8/1.6/0.32/0ng/ml with a diluent (1% BSA-PBST), and the resultant mixture was sequentially added to the enzyme-labeled plate (blocked), 100. Mu.l/well, and left at 37 ℃ for 1 hour. PBST washing plate 3 times, adding HRP labeled goat anti-mouse IgG secondary antibody (purchased from Millipore, cat. AP 181P), standing at 37 deg.C for 30min; after PBST washing for 5 times, residual droplets were patted dry on absorbent paper, 100. Mu.l of TMB (from BD Co., cat. No. 555214) was added to each well, and the mixture was left for 5min at room temperature (20. + -. 5 ℃) in the dark; add 50. Mu.l 2M H per well ₂ SO ₄ And stopping the substrate reaction by the stop solution, reading the OD value at 450nm of the microplate reader, and analyzing the binding capacity of the antibody to be detected and the target antigen IL-2.

As shown in FIG. 1, the murine anti-human IL-2 monoclonal antibody m22F8 bound human IL-2 with an EC50 of 6.90ng/ml, i.e., 0.05nM, showing good affinity.

Example 4 murine anti-human IL-2 monoclonal antibody m22F8 blocks human IL2 binding to CD25-ECD

Recombinant hCD25 (amino acid sequence shown in SEQ ID NO: 2) was diluted to 1. Mu.g/ml with a coating solution (50 mM carbonate coating buffer, pH 9.6), added to an ELISA plate at 100. Mu.l/well, and coated overnight at 4 ℃. PBST washing plate 3 times, adding 200 u l/hole blocking solution (2% BSA-PBST), 37 degrees C placed 1h PBST washing plate 1 times for use. Mouse-derived anti-human IL-2 monoclonal antibody m22F8After dilution (1% BSA-PBST) was diluted in a gradient to 10000/2000/400/80/16/3.2/0.64/0ng/ml, the mixture was mixed with bio-IL2 diluted to 20ng/ml in the same volume, incubated at 37 ℃ for 30min, added to the blocked ELISA plate at 100. Mu.l/well, and left at 37 ℃ for 1h. PBST washing plate 3 times, adding HRP-labeled SA (SA-HRP, pierce company), standing at 37 deg.C for 30min; after PBST washing for 5 times, residual droplets were patted dry on absorbent paper, 100. Mu.l of TMB (from BD Co., cat. No. 555214) was added to each well, and the mixture was left for 5min at room temperature (20. + -. 5 ℃) in the dark; add 50. Mu.l of 2MH per well ₂ SO ₄ And stopping the substrate reaction by the stop solution, reading an OD value at 450nm of the microplate reader, and analyzing the effect of the to-be-detected antibody on blocking IL-2 from being combined with CD 25.

As shown in FIG. 2, the murine anti-human IL-2 monoclonal antibody m22F8 was able to effectively inhibit IL-2 binding to CD25 with an IC50 of 253.1ng/ml, i.e., 1.69nM.

Example 5 growth of MC38 cell transplantable tumors inhibited by IL-2/m22F8 Complex in mice

Collecting mouse colon cancer MC38 cells cultured in vitro, adjusting cell suspension concentration to 1 × 10 ⁷ And (4) the concentration is/ml. C57BL/6 mice were shaved on the right flank. Under sterile conditions, 100. Mu.l of cell suspension was inoculated subcutaneously into the right flank of C57 mice. Measuring the diameter of the subcutaneous transplanted tumor of the mouse by using a vernier caliper until the average tumor volume grows to 100-200mm ³ Animals were then randomly assigned to groups of 8 animals per group. IL-2 and m22F8 are mixed according to the mass ratio of 1:7, and after incubation for 15 minutes at room temperature, an IL-2/m22F8 compound is prepared, wherein the compound is dosed according to 1mg/kg of IL-2, an equal amount of PBS is dosed to a control group, and the compound is dosed by intraperitoneal injection for 2 times every week and is dosed for 2 times continuously. Throughout the experiment, the diameter of the transplanted tumor was measured 2 times per week, and the body weight of the mice was weighed. The formula for Tumor Volume (TV) is:

TV＝1/2×a×b2

wherein a and b represent length and width, respectively. Calculating Relative Tumor Volume (RTV) according to the measurement result, wherein the calculation formula is as follows: RTV = Vt/V0. Where V0 is the tumor volume measured at the time of group administration (i.e., d 0) and Vt is the tumor volume at each measurement. The evaluation index of the antitumor activity is TGI (tumor inhibition ratio%) T/C (%), and the calculation formula is as follows:

TGI％＝100％-T/C(％)

relative tumor proliferation rate T/C (%) = (TRTV/CRTV) × 100

TRTV: treatment group RTV; CRTV: negative control group RTV.

The results are shown in FIG. 3, the IL-2/m22F8 compound shows excellent antitumor activity, and the tumor inhibition rate is close to 100% after 2 times of administration. However, in the experimental process, the animals generally have clinical symptoms including reduced activity, reduced feeding, loose hair, reduced body temperature and the like, and 2/8 of the animals die. It is demonstrated that the IL-2/m22F8 complex, although having good in vivo anti-tumor effects, has safety problems, including the appearance of significant clinical symptoms, and even death.

Example 6 determination of mouse hybridoma antibody Gene and preparation of chimeric antibody

This example obtained the heavy chain variable region and the light chain variable region of hybridoma m22F8 by a related method of molecular biology and further used for constructing a chimeric antibody.

The RNA of hybridoma cells was extracted by Trizol and subjected to mRNA reverse transcription to obtain cDNA, followed by PCR using cDNA as a template and degenerate primers for heavy and light chains of murine Antibody (the sequences of the combined primers come from page 323) respectively, sequencing the obtained PCR product and determining the obtained sequence as the variable region sequence of murine Antibody by Kabat database analysis.

The relevant sequence information is as follows:

m22F8 heavy chain variable region gene sequence, the total length is 351bp, 117 amino acid residues are coded, the nucleotide sequence is shown as SEQ ID NO. 3, and the amino acid is shown as SEQ ID NO. 4; the full length of the m22F8 monoclonal antibody light chain variable region gene sequence is 318bp, 106 amino acid residues are coded, the nucleotide sequence is shown as SEQ ID NO. 8, and the amino acid sequence is shown as SEQ ID NO. 9.

Recombining each obtained hybridoma heavy chain variable region sequence with a human IgG4 constant region (containing S228P mutation) (the amino acid sequence is shown as SEQ ID NO: 13) to form a chimeric ch22F8 monoclonal antibody heavy chain (the amino acid sequence is shown as SEQ ID NO: 15); the light chain variable region sequence was recombined with the human kappa chain constant region (amino acid sequence shown in SEQ ID NO: 14) to form the chimeric ch22F8 mab light chain (amino acid sequence shown in SEQ ID NO: 16).

The heavy chain variable region (amino acid sequence shown in SEQ ID NO: 19) and the light chain variable region (amino acid sequence shown in SEQ ID NO: 20) of NARA1 mAb were synthesized according to literature (patent US 2017/0183403 Al). The heavy chain variable region of the NARA1 monoclonal antibody is recombined with the human IgG4 constant region to form the NARA1 monoclonal antibody heavy chain (the amino acid sequence is shown in SEQ ID NO: 21), and the light chain variable region of the NARA1 monoclonal antibody is recombined with the human kappa chain constant region to form the NARA1 monoclonal antibody light chain (the amino acid sequence is shown in SEQ ID NO: 23).

The heavy chain and light chain genes are respectively constructed to pcDNA3.4 expression vectors, are matched and transfected to Expi-293F cells, chimeric antibodies ch22F8 and NARA1 are obtained through Protein A purification, the molecular weight of each expressed antibody is determined to be about 150kD through SDS-PAGE electrophoresis and SEC-HPLC, the purity of the antibody is more than 95 percent, and the antibody is quantified, subpackaged and frozen at-80 ℃ for later use.

Example 7 preparation of anti-human IL-2 chimeric antibody

Adopting gene site-directed mutagenesis technology or gene synthesis technology, converting the 310 th site H of a human IgG4 constant region (the amino acid sequence is shown as SEQ ID NO: 13) into A and the 435 th site amino acid H into Q, keeping other amino acid sequences unchanged to form a mutated human IgG4 constant region (the amino acid sequence is shown as SEQ ID NO: 17), recombining the m22F8 heavy chain variable region with the mutated human IgG4 constant region to form an anti-human IL-2 chimeric antibody heavy chain (the amino acid sequence is shown as SEQ ID NO: 18); the heavy chain variable region of NARA1 monoclonal antibody recombines with the mutated human IgG4 constant region to form the mutated NARA1 (NARA 1 mu) heavy chain (the amino acid sequence is shown in SEQ ID NO: 22).

The mutant heavy chain genes are respectively constructed to pcDNA3.4 expression vectors, the respective light chains prepared in the example 6 are transfected into Expi-293F cells, anti-human IL-2 chimeric antibody (ch 22F8 mu) and NARA1mu are obtained by Protein G purification, the molecular weight of each expressed antibody is about 150kD and the antibody purity is more than 95 percent determined by SDS-PAGE and SEC-HPLC, and the antibodies are quantified, subpackaged and frozen at-80 ℃ for later use.

Example 8 lethality of IL-2/ch22F8mu Complex to Experimental mice

IL-2 was mixed with each test antibody (ch 22F8, ch22F8mu, NARA1, NARA1 mu) at a mass ratio of 1:7, incubation for 15 minutes at room temperature, forming a complex, 1mg/kg and/or 3mg/kg and/or 6mg/kg dose (see table 1 in particular) of IL-2, i.p. injecting C57BL/6 mice (Wintoli Hua) 2 times on

days

1 and 4, observing the death of the experimental mice on day 7, and administering PBS in the same volume to the control group.

TABLE 1 administration of the respective antibodies

Dosage (IL-2)	ch22F8	ch22F8mu	NARA1	NARA1mu
					1mg/kg	√	-	-	-
3mg/kg	√	√	√	√
					6mg/kg	-	√	-	-

TABLE 2 survival rates of experimental animals for each antibody dose group

Antibodies	1mg/kg	3mg/kg	6mg/kg
				IL-2/ch22F8	40％	0％	-
IL-2/ch22F8mu	-	100％	100％
				NARA1	-	0％	-
NARA1mu	-	100％	-

The results are shown in table 2:

(1) On the 7 th day of experiment, all experimental animals in the ch22F8 and NARA1 groups died (survival rate of 0%) but all experimental animals in the ch22F8mu and NARA1mu groups survived (survival rate of 100%) at the dose of 3mg/kg, which shows that the toxicity of the IL-2 monoclonal antibody to the experimental mice is obviously reduced by the compound formed by IL-2 after the mutation.

(2) The survival rate of the experimental animals in the ch22F8mu group is still 100% when the dosage is increased to 6mg/kg, and 60% of the animals die when the dosage of the ch22F8 is reduced to 1mg/kg, which further indicates that the toxicity of the complex formed by the ch22F8mu and IL-2 to the experimental mice is obviously reduced after the Fc mutation. Example 9 IL-2/ch22F8mu reduces liver damage in Experimental animals

IL-2 was mixed with the test antibodies ch22F8mu and ch22F8, respectively, at a mass ratio of 1:7, and incubated at room temperature for 15 minutes to form a complex, at a dose of 3mg/kg, based on IL-2, by intraperitoneal injection of C57BL/6 mice (Wintoli Hua Co.) 2 times on

days

1 and 4; the dose of 1mg/kg based on IL2 was 2 times per week, C57BL/6 mice (Wintolite Co.) were intraperitoneally injected 6 times, and the serum was obtained from the blood of the surviving mice the next day after the last administration, and ALT was determined.

Results are shown in FIGS. 4 and 5:

(1) At IL-2 dose of 1mg/kg, 3/8 mice in IL-2/ch22F8 group died, with an ALT average of 425.2U/L in surviving 5/8 mice, and no animals in IL-2/ch22F8mu group, and an ALT average of 64.0U/L in all 8 mice.

(2) At IL-2, 3mg/kg IL-2/ch22F8 group, 3/8 mice died, with surviving mice ALT averaging 303.2U/L, IL-2/ch22F8mu group dead animals, and all 8 mice ALT averaging 62.3U/L.

(3) The mean ALT value for the PBS control group was 29.4U/L, indicating that IL-2/22F8mu had significantly less hepatotoxicity than IL-2/ch22F 8.

Example 10 in vivo antitumor Activity of IL-2/ch22F8mu Complex

Experimental method As in example 5, IL-2 was mixed with each test antibody (ch 22F8, ch22F8 mu) at a mass ratio of 1:7, and incubated at room temperature for 15 minutes to form a complex, wherein the doses of 0.3mg/kg and 1mg/kg were measured in terms of IL-2, and the dose of 1mg/kg was measured as a single IL-2 drug as a control. The administration is performed 3 times per week by intraperitoneal injection for 2 weeks.

As shown in FIG. 6, both the control IL-2 single drug and the control ch22F8mu single drug showed no antitumor effect, and at the dose of IL-2 0.3mg/kg, both the IL-2/ch22F8 and IL-2/ch22F8mu groups showed weak activity, with TGI of 24.6% and 38.2%, respectively; under the dosage of 1.0mg/kg calculated by IL-2, the IL-2/ch22F8 and IL-2/ch22F8mu groups both show strong activity, the TGI is 72.9 percent and 71.9 percent respectively, and no significant difference exists, which indicates that the IL-2/ch22F8mu retains good in-vivo anti-tumor activity.

Example 11 preparation of humanized anti-human IL-2 monoclonal antibody and humanized anti-human IL-2 chimeric antibody

The amino acid sequences of the light chain variable region and the heavy chain variable region of the candidate murine antibody of example 1 were analyzed to determine the 3 antigen Complementarity Determining Regions (CDRs) and 4 Framework Regions (FRs) of the murine antibody according to Kabat's rules. The amino acid sequence of the 22F8 heavy chain complementarity determining region is HCDR1: GFNIKNTY (amino acid sequence shown in SEQ ID NO: 5), HCDR2: IDPANGNT (amino acid sequence is shown in SEQ ID NO: 6), HCDR3: GRSRGYAMDY (amino acid sequence shown in SEQ ID NO: 7), and the amino acid sequence of the light chain complementarity determining region is LCDR1: DHINNW (amino acid sequence shown in SEQ ID NO: 10), LCDR2: GATSLET (amino acid sequence shown in SEQ ID NO: 11) and LCDR3: QQYWSTPT (amino acid sequence shown in SEQ ID NO: 12).

The humanized template that best matches the non-FR region of each murine antibody described above was selected in the Germine database. Then, the CDR region of the murine antibody was grafted onto a selected humanized template, the CDR region of the humanized template was replaced, the heavy chain variable region was recombined with the human IgG4 constant region (containing the S228P mutation), the light chain variable region was recombined with the human kappa chain constant region, and simultaneously, based on the three-dimensional structure of the antibody, the buried residues, the residues that directly interact with the CDR region, and the residues that significantly affect the conformation of VL and VH of each antibody were subjected to back mutation to finally obtain the humanized anti-human IL-2 monoclonal antibody (hu 22F 8) heavy chain variable region (amino acid sequence shown in SEQ ID NO: 24), which was recombined with the human IgG4 constant region to obtain the recombinant humanized anti-human IL-2 monoclonal antibody (amino acid sequence shown in SEQ ID NO: 27), the human anti-human IL-2 antibody (hu 22F8 mu) heavy chain (amino acid sequence shown in SEQ ID NO: 26), the humanized anti-human IL-2 monoclonal antibody (amino acid sequence shown in SEQ ID NO: 25), and the human kappa chain constant region were recombined to obtain the human IL-human heavy chain variable region (amino acid sequence shown in SEQ ID NO: 28). Respectively constructing heavy chains and light chains of the humanized antibodies to pcDNA3.4 expression vectors, transfecting Expi-293F cells, purifying through Protein G to obtain humanized anti-human IL-2 monoclonal antibodies (hu 22F 8) and mutated humanized anti-human IL-2 antibodies (hu 22F8 mu), and determining that the molecular weight of each antibody is correct and the purity is more than 95% through SDS-PAGE electrophoresis and SEC-HPLC.

Example 12 ch22F8mu and hu22F8mu bind to human IL-2

Each monoclonal antibody was detected to bind to human IL-2 by ELISA as in example 3.

As shown in FIG. 7, the EC50 for ELISA detection of hu22F8 and hu22F8mu binding to human IL-2 were: the EC50 s for binding of human IL-2 by 6.76ng/ml and 6.01ng/ml, i.e., 0.05nM and 0.04nM, ch22F8 and ch22F8mu, respectively, are: 6.29ng/ml and 7.24ng/ml, i.e. 0.04nM and 0.05nM.

Example 13, hu22F8mu and ch22F8mu block IL-2 binding to CD25

The experimental procedure was as in example 4.

As shown in FIG. 8, the IC50 for hu22F8 and hu22F8mu, respectively, for blocking human IL-2 binding to CD25 are: 218.6ng/ml and 227.9ng/ml, i.e., 1.46nM and 1.52nM; the IC50 for ch22F8 and ch22F8mu to block human IL-2 binding to CD25 were: 256.7ng/ml and 236.6ng/ml, i.e., 1.71nM and 1.58nM. It was shown that the above-mentioned mutated humanized or chimeric antibody of Fc did not affect its effect of blocking the binding of IL-2 to CD 25.

Example 14 IL-2/hu22F8mu Complex (SPGD 01) stimulates CTLL2 cell proliferation

This example demonstrates the in vitro biological activity of the complexes IL-2/hu22F8mu (SPGD 01), IL-2/hu22F8, IL-2/ch22F8 and IL-2/ch22F8mu in a CTLL2 cell proliferation assay. The method comprises the following steps:

CTLL2 cells were diluted to 1E5/ml in 10% FBS-containing 1640 medium and added to the cell culture plate at 100 ul/well. IL-2 was mixed with hu22F8mu, hu22F8, ch22F8 and ch22F8mu, 1:7, respectively, and left at room temperature for 30 minutes to form complexes SPGD01 (IL 2/hu22F8 mu), IL-2/hu22F8, IL-2/ch22F8 and IL-2/ch22F8mu, IL-2 and each of the above complexes was diluted to 50ng/ml in a 10 FBS-containing 1640 culture solution in terms of IL-2, and the above CTLL 2-containing culture plates were added after 8 gradients of 2-fold dilution, 37 ℃ and 5 CO were added to each of the above CTLL 2-containing culture plates ₂ After the cells were cultured in a cell incubator for 72 hours and diluted, the relative cell count of each well was measured by CCK8, and the EC50 was calculated to determine the activity of the sample.

As shown in FIG. 9, IL-2, SPGD01 (IL-2/hu 22F8 mu), IL-2/hu22F8, IL-2/ch22F8 and IL-2/ch22F8mu all stimulated proliferation of CTLL2 cells, with an EC50 of IL-2 of 0.74ng/ml, an EC50 of SPGD01, IL-2/hu22F8 of 1.01ng/ml and 1.09ng/ml, respectively, and an EC50 of IL-2/ch22F8, IL-2/ch22F8mu of 0.98ng/ml and 1.04ng/ml, respectively, indicating that SPGD01 and IL-2/hu22F8 have consistent biological activity, and IL-2/ch22F8 also have biological activity, further indicating that Fc mutation does not result in the biological activity of IL-2/antibody complexes.

Example 15, the ability of SPGD01 to bind FcRn is significantly reduced

SPGD01 (IL-2/hu 22F8 mu) and IL-2/hu22F8 were tested for binding to human FcRn/β 2M (purchased from Beijing Yiqiao Shenzhou, cat # CT 009-H08H) and mouse FcRn/β 2M (purchased from Beijing Yiqiao Shenzhou, cat # CT 029-M08H) by ELISA methods, as described in example 3. After mouse FcRn/beta 2M and human FcRn/beta 2M are blocked by an enzyme label plate at 1 ug/Kong Bao, the enzyme label plate is added after being diluted by PBS with pH 6.0 to SPGD01 or IL2/hu22F8 to 10ug/ml (based on the mass of the antibody) and diluted by 5 times of ratio gradient, and a buffer system with pH 6.0 is adopted in the whole experiment process, and the process is the same as that of the embodiment 3.

As shown in FIG. 10, SPGD01 (IL 2/hu22F8 mu) bound human FcRn/β 2M significantly less than IL2/hu22F8, and overall, both SPGD01 and IL2/hu22F8 bound human FcRn/β 2M less strongly.

As shown in FIG. 11, SPGD01 (IL 2/hu22F8 mu) bound murine FcRn/β 2M significantly less than IL2/hu22F8, and overall, both SPGD01 and IL2/hu22F8 bound murine FcRn/β 2M less strongly.

Example 16 reduction of lethality of SPGD01 (IL-2/hu 22F8 mu) in Experimental mice

The experimental procedure was as in example 8.

As shown in Table 3, at the dose of 3mg/kg, 100% (10/10) of the experimental animals died in the IL-2/hu22F8 group, and none of the experimental animals died in the SPGD01 (IL-2/hu 22F8 mu) group (0/10); 50% of the experimental animals survived the IL-2/hu22F8 group at a dose of 1 mg/kg; and the SPGD01 group survived 100% of the experimental animals at high dose up to 6mg/kg, which shows that the toxicity of the complex formed by the antibody Fc and IL-2 to the experimental mice is obviously reduced after the mutation, and the result is consistent with IL2/ch22F8 mu.

TABLE 3 survival rates of experimental animals for each antibody dose group

Antibodies	1mg/kg	3mg/kg	6mg/kg
				IL-2/hu22F8	50％	0％	-
IL-2/SPGD01	-	100％	100％

Example 17 in vivo antitumor Activity of SPGD01 (IL-2/hu 22F8 mu)

The experimental procedure was as in example 5.

The results are shown in FIG. 12, the IL-2/hu22F8 complex showed good antitumor activity, with TGI reaching 95.4% and 85.6% in the 0.5mg/kg and 0.25mg/kg groups, respectively; SPGD01 (IL-2/hu 22F8 mu) also exhibited excellent antitumor activity with TGI reaching 95.8%, 84.5% and 75.4% for the 2mg/kg,1mg/kg and 0.5mg/kg groups, respectively. Statistical analysis shows that the two have no significant difference in antitumor effect (p > 0.05).

In the experimental process, animals die after the 1mg/kg dose group of IL2/hu22F8 is administrated for 2 times, and no animals die after the other groups are administrated for 6 times, which shows that the IL2/hu22F8 compound has stronger toxicity, and the safety of SPGD01 (IL-2/hu 22F8 mu) is obviously improved after Fc mutation.

Example 18 synergistic antitumor Activity of SPGD01 (IL-2/hu 22F8 mu) with PD1 mAb in vivo

The MC38 transplantation tumor model is adopted to evaluate the in vivo synergic anti-tumor effect of SPGD01 (IL-2/hu 22F8 mu) and anti-PD 1 monoclonal antibody, and the experimental method is the same as that in example 5.SPGD01 dose was 4mg/kg as IL-2, and rat anti-mouse PD1 mab (purchased from Bio X Cell, cat # BP 0146) was 5mg/kg, administered by intraperitoneal injection 3 times/week for 6 times. 3 days after the last administration, the mice were sacrificed to obtain mouse serum and mouse spleen, the glutamic-pyruvic transaminase (ALT) content in the mouse serum was determined by the enzyme activity method, and the spleen cells, CD4 positive cells, CD8 positive cells, and CD4 and CD25 positive cells were analyzed by the conventional flow method (FACS). Other reference is made to example 5.

The results are shown in fig. 13, the SPGD01 (IL 2/hu22F8 mu) single drug and the PD1 mab (anti-mPD 1) single drug both showed moderate antitumor effects, with TGIs of 61.7% and 41.3% respectively, and the TGI of the combined drug reached 87.8%, showing good synergistic effects.

As shown in FIG. 14, there was no significant difference in ALT values between the administered groups and the PBS control group (p > 0.05).

Results as shown in fig. 15, the CD 4-positive cell to CD 8-positive cell ratio (CD 4/CD 8) was not significantly different from that of PBS group (p > 0.05) in anti-mPD1 group, but the SPGD01 group and combination group (SPGD 01+ anti-mPD 1) group were significantly lower than PBS group by a very significant difference (p <0.01, p and 0.001).

The results are shown in fig. 16, the ratio of CD4+ CD25+ to CD3 positive cells (CD 4+ CD25+/CD3 +) was not significantly different from PBS (p > 0.05) in the anti-mPD1 group, but significantly different (p < 0.05) between SPGD01 and combination (SPGD 01+ anti-mPD 1) groups than PBS group.

Example 19, SPGD01 (IL-2/hu 22F8 mu) and hu22F8mu have good stability

IL-2 and hu22F8mu are uniformly mixed according to the mass ratio of 1:7 to form SPGD01 (IL-2/hu 22F8 mu), the SPGD01 and hu22F8mu are placed at 4 ℃ for a corresponding time, the change of purity of the SPGD01 and hu22F8mu after being placed for different times is detected by using molecular sieve high performance liquid chromatography (SEC-HPLC), and the stability of the SPGD01 and hu22F8mu is examined. The method comprises the following steps:

the reaction was carried out on an HPLC Ultimate 3000 (Thermo) chromatograph using a TSKgel G3000SWXL column (TSK Co.). The mobile phase was detected as PBS (pH 7.4), at a constant flow rate of 0.8ml/min, and the loading was 100ug/100ul. Purity was expressed by calculating the content (%) of the target protein in the total protein by the 280nM absorbance peak integration method.

As shown in FIGS. 17, 18, 19 and 20, SPGD01 (IL-2/hu 22F8 mu) was 95% pure in PBS solution at 0 weeks, 94% pure after 24 weeks at 4 ℃ and a change rate of <2%; the purity of the monoclonal antibody hu22F8mu in PBS solution was 93% at 0 weeks and 95% after 6 weeks at 4 ℃, with a <3% change rate, indicating that the IL-2/antibody complex SPGD01 and the related monoclonal antibody hu22F8mu remain stable in PBS buffer at 4 ℃.

The foregoing embodiments are merely illustrative of the principles of the present invention and its efficacy, and are not to be construed as limiting the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. An anti-human IL-2 monoclonal antibody having:

the heavy chain variable region having the amino acid sequence of SEQ ID NO. 4 and the light chain variable region having the amino acid sequence of SEQ ID NO. 9.

2. The nucleic acid encoding the anti-human IL-2 monoclonal antibody of claim 1, which has:

3. An anti-human IL-2 chimeric antibody, which has:

4. An Fc-mutated anti-human IL-2 chimeric antibody, characterized in that it has:

a heavy chain sequence having the amino acid sequence of SEQ ID NO. 18, and a light chain sequence having the amino acid sequence of SEQ ID NO. 16.

5. A humanized anti-human IL-2 monoclonal antibody, which is constructed by using CDRs grafting technology and CDR mutation design on the basis of the anti-human IL-2 monoclonal antibody of claim 1.

6. The humanized anti-human IL-2 monoclonal antibody of claim 5, which has: the heavy chain variable region having the amino acid sequence of SEQ ID NO. 24 and the light chain variable region having the amino acid sequence of SEQ ID NO. 25.

7. A humanized anti-human IL-2 antibody having:

the heavy chain sequence having the amino acid sequence of SEQ ID NO. 27, and the light chain sequence having the amino acid sequence of SEQ ID NO. 28.

8. An Fc-mutated humanized anti-human IL-2 antibody having:

9. An antibody complex, which is prepared by mixing IL-2 with the anti-human IL-2 chimeric antibody of claim 3 or the Fc-mutated anti-human IL2 chimeric antibody of claim 4 or the humanized anti-human IL-2 antibody of claim 7 or the Fc-mutated humanized anti-human IL-2 antibody of claim 8 at a mass ratio of 1:7.

10. Use of an antibody complex according to claim 9 in the preparation of a medicament for the treatment of an antineoplastic agent.