Patent Application
20250179208
The present invention relates to an antibody or antigen-binding fragment that binds to human CD26 and its application in the preparation of drugs for treating tumors with high expression of CD26.
CD26 is a multifunctional type II transmembrane glycoprotein that can also be found in plasma in a dissolved form. CD26 usually exists as a homodimer with a monomer including 766 amino acids and a relative molecular mass of about 110 kDa. The amino acid residues of CD26 can be divided into five parts from inside to outside: intracellular region (1-6), transmembrane region (7-28), highly glycosylated region (29-323), cysteine-rich region (324-551) and C-terminal catalytic domain (552-766). The molecular structure of CD26 is closely related to its function. CD26 (DPP4) inhibitors have been used in the treatment of type 2 diabetes for decades. The expression of CD26 is significantly increased on the surface of a variety of tumor cells, such as malignant mesothelioma, renal cancer, prostate cancer, lung cancer, etc. CD26 is a valuable target for these types of tumors with high CD26 expression (CD26/DPP4-a potential biomarker and target for cancer therapy, Pharmacology & Therapeutics (2019), 198:135-159).
At present, the most advanced anticancer drug research targeting human CD26 is the monoclonal antibody YS110 from Y's Therapeutics, which has completed phase I/II clinical trials. However, according to the existing literature, YS110 has been found to have low activity in preclinical studies both in vitro and in vivo. As in the literature “A humanized anti-CD26 monoclonal antibody inhibits cell growth of malignant mesothelioma via retarded G2/M cells In cycle transition, Cancer Cell Int (2016), 16:35”, after treated with 250 μg/ml YS110 for 48 h, the growth inhibition rate of tumor cell line was 18.3%, and the IC50 value was much higher than 250 ug/ml. These results indicated that its activity in vitro was low. In the patent “anti-CD26 antibody and use thereof” (Application No. CN200680034937.4), after the treatment of YS110 in mice bearing a variety of CD26 high expression tumor cell lines, YS110 can only reduce tumor volume and alleviate tumor growth to a certain extent, but can not completely inhibit tumor growth to achieve tumor cure, showing its activity in animal models is still not ideal. Results from the completed phase 2 clinical study of YS110 have shown that YS110 is well tolerated, but disease control rates have not been as high as expected, which corresponds to the low activity of YS110 in preclinical studies.
Although the widespread distribution of CD26 has been reported in the literature, the use of YS110 is still safe, indicating that its safety in clinical use is well guaranteed. In the 31 patients of YS110 clinical trial, there was no patient death. The main side effects were infusion adverse reactions (16.1%), hiccups (9.7%) and diarrhea (6.5%). (Phase 2 Study of YS110, a Recombinant Humanized Anti-CD26 Monoclonal Antibody, in Japanese Patients With Advanced Malignant Pleural Mesothelioma, JTO Clinincal and Research Reports (2021), 2 (6).)
In conclusion, although the safety of CD26 has been verified in clinical trials, the existing antibodies targeting CD26 are difficult to achieve good therapeutic effects on tumor treatment.
To solve the above problems, the first aim of the present invention is to provide a more effective and safer CD26 antibody or antigen-binding fragment.
An antibody or antigen-binding fragment specifically binding to human CD26 of the present invention includes HCDR1 shown as SEQ ID NO:1, HCDR2 shown as SEQ ID NO:2, and HCDR3 shown as SEQ ID NO:3; as well as LCDR1 shown as SEQ ID NO:4, LCDR2 consisting of Arg Met Ser, and LCDR3 shown as SEQ ID NO:5.
The antibody or antigen-binding fragment includes a variable region of heavy chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8, and a variable region of light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9.
The antibody or antigen-binding fragment, which is one, two, three, four, five, six, seven, eight, nine, or ten amino acids of the sequence shown as SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, or SEQ ID NO:9 are inserted, deleted, or substituted. The amino acid substitutions are conservative amino acid substitutions.
The antibody or antigen-binding fragment includes a variable region of heavy chain shown as SEQ ID NO:6 and a variable region of light chain shown as SEQ ID NO:7; Or a variable region of heavy chain shown as SEQ ID NO:8 and a variable region of light chain shown as SEQ ID NO: 9.
The second purpose of the present invention is to provide a bispecific T cell engager (BiTE) including the sequence of an antibody or antigen-binding fragment targeting CD26.
The bispecific T cell engager (BiTE) including the sequence of an antibody or antigen-binding fragment targeting CD26 of the present invention, is formed by connecting two antigen-specific single-chain variable fragments (scFv) through a linker. One scFv targeting CD26, is formed by connecting a variable region of heavy chain and a variable region of light chain targeting CD26 through a linker; Another scFv targets CD3, is formed by connecting a variable region of heavy chain and a variable region of light chain targeting CD3 through a linker; The scFv targeting CD26 includes HCDR1 shown as SEQ ID NO:1, HCDR2 shown as SEQ ID NO:2, HCDR3 shown as SEQ ID NO:3; As well as LCDR1 shown as SEQ ID NO:4, LCDR2 consisting of Arg Met Ser, and LCDR3 shown as SEQ ID NO:5.
The scFv targeting CD26 includes a variable region of heavy chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO:8, and a variable region of light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9.
The scFv targeting CD26 includes a sequence wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids of the sequence shown as SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9 are inserted, deleted, or substituted. The amino acid substitutions are conservative amino acid substitutions.
The scFv targeting CD26 includes a variable region of heavy chain shown as SEQ ID NO: 6 and a variable region of light chain shown as SEQ ID NO:7; or includes a variable region of heavy chain shown as SEQ ID NO:8 and a variable region of light chain shown as SEQ ID NO: 9.
The scFv targeting CD3 is derived from OKT-3, L2K, TR66, UCHT1, SP34, IORT3, Catumaxomab, Blinatumomab, Solitomab and other CD3 antibody sequences known in the art. Such as the anti-CD3 variable region of heavy chain shown as SEQ ID NO:10 and the anti-CD3 variable region of light chain shown as SEQ ID NO:11.
The linker connecting the variable region of heavy chain and the variable region of light chain in scFv is selected from the linkers commonly used in the art. For example, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSTSGGGSGGGGSS, GSTSGSGKPGSGEGSTKG, (GGGGS)n, where n is an integer from 1 to 5, preferably n is an integer from 1 to 3, preferably n is 3, shown as SEQ ID NO: 12.
The linker connecting two scFvs is selected from linkers commonly used in the art, such as (GGGGS)n, where n is an integer from 1 to 5, preferably an integer from 1 to 3, preferably n is 1, shown as SEQ ID NO:13.
As preferred, the bispecific T cell engager including the sequence of an antibody or antigen-binding fragment targeting CD26 is shown as SEQ ID NO:14 (amino acid sequence of 18G272 BiTE) or SEQ ID NO:15 (amino acid sequence of 19G294 BITE).
The invention also relates to a nucleotide sequence encoding an amino acid sequence of the antibody or antigen-binding fragment. The invention also relates to a vector including the nucleic acid, a host cell including the nucleic acid.
The present invention also relates to methods for producing antibodies or antigen-binding fragments, including culture of the host cell, recovery of the antibody or antigen-binding fragments from the culture.
The present invention also relates to a pharmaceutical composition including the antibody or antigen-binding fragment, which also includes a pharmaceutically acceptable excipient.
The present invention also relates to a method of treating tumors, including administering to a patient an effective amount of the antibody or antigen-binding fragment; The tumors highly expressing CD26, includes but not limited to renal cell carcinoma, mesothelioma, lung cancer, liver cancer, prostate cancer, etc. The antibody or antigen-binding fragment is administered alone or in combination with other anticancer agents.
First, the Mouse-Derived Antibodies Screened in this Application have Obvious Advantages.
Example 1 shows that No. 62 mouse derived antibody screened in this application has a much higher lysis intensity to human tumor cells than other mouse derived antibodies. Moreover, the killing effect of the mouse derived antibody screened in this application on human tumor cells has been equivalent to that of the humanized antibody YS110 which has completed the clinical phase II, which fully proves the advantages of the mouse derived antibody.
In addition, the data of examples 5, 8 and 10 show that the chimeric antibody or humanized antibody based on the variable region of No. 62 mouse antibody has better dot blot activity and better affinity with antigen than the corresponding antibody derived from other mouse antibodies.
Compared with BiTE derived from other mouse derived antibodies, the BiTE based on the variable region of No. 62 mouse antibody in this application shows better effectiveness. Example 11 shows when comparing the BiTE mediated cytotoxicity of PBMC to different target cells in vitro, the BiTE derived from No. 62 mouse antibody of the application has better killing effect on a variety of tumor cells than the BiTE derived from CD26 antibody YS110. The results of Examples 17-22 show that the proposed BiTE can significantly inhibit tumor growth in a variety of animal tumor models, and can completely inhibit tumor growth in some models, which is significantly better than the BiTE derived from YS110.
Furthermore, the BiTE based on the variable region of No. 62 mouse antibody of this application shows better safety (Example 13) and a lower risk of causing cytokine storm than BiTE derived from other mouse antibody.
Second, the Proposed Bispecific T Cell Engager Targeting CD26 is More Effective and Safer than Available Full-Length Monoclonal Antibodies Targeting CD26.
In terms of effectiveness, the data of Example 11 show that BiTE of this application has a better killing effect on a variety of CD26 positive tumor cells than the full-length IgG antibody YS110. The data from Examples 23 and 24 show that the BiTE of this application has significantly better tumor growth inhibition than the full-length IgG antibody YS110 in a variety of animal tumor models.
In terms of safety, the data of Example 12 show that anti-CD26 full-length IgG antibody does not effectively induce T cell activation. The BiTE of this application does not cause significant T cell activation before reaching the tumor site. After binding to the tumor cells, the BiTE of this application can cause the activation of T cells around the tumor tissue, and then kill the tumor cells, and improve the safety. The data from examples 23 and 24 show that the BiTE of this application shows significantly better safety than the full-length IgG antibody YS110 in a variety of animal tumor models.
Third, the Bispecific T Cell Engager Targeting CD26 in the Present Application Shows Superior Efficacy and Safety Compared to Existing First-Line Treatment Options for Related Tumors, Such as BAVENCIO for Renal Cancer (Examples 15, 22, 23).
Unless clearly defined herein, technical terms used herein have meanings generally understood by one of ordinary skill in the art.
Singular words such as “a, an” and “the” include their corresponding plural form.
The term “or” means “and/or” and can be used interchangeably with “and/or”.
The term “CD26” is also known as DPP4 (dipeptidyl peptidase 4). The amino acid sequence of human CD26 can be found in Genbank with accession number NP_001926.2. Its cDNA sequence can be found in Genbank with the accession number NM_001935.3.
The term “conservative amino acid substitution” means the replacement of an original amino acid with a new amino acid that substantially does not change the chemical, physical, and/or functional properties of the antibody or fragment, such as its binding affinity to CD26. Common conserved substitutions of amino acids are well known in the art, for example, Ala can be conservatively substituted with Gly or Ser; Arg could be conservatively replaced by Lys or His. Asn can be conservatively replaced by Gln, His, etc. The term “affinity” refers to the strength of the interaction between an antibody and an antigen. The variable region of antibody interacts with antigen by non-covalent force. The more interactions, the stronger the affinity.
The term “antibody” refers to a family of immunoglobulins that can specifically bind the corresponding antigen noncovalently, reversibly. For example, naturally IgG antibodies are tetramers that includes at least two heavy chains and two light chains linked to each other by disulfide bonds. Each heavy chain consists of a variable region of heavy chain (VH) and a constant region of heavy chain (CH). The constant region of heavy chain consists of three domains CH1, CH2, and CH3. Each light chain consists of a variable region of light chain (VL) and a constant region of light chain (CL). The constant region of light chain consists of one domain, CL. VH and VL can be further subdivided into complementarity determining regions (CDR, also known as hypervariable regions) with high variability, and a more conserved framework region (FR). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from amino-terminal to carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. HCDR1, HCDR2 and HCDR3 are three complementarity determining regions of heavy chain, and LCDR1, LCDR2 and LCDR3 are three complementarity determining regions of light chain. The location of the CDR and framework region may be determined using various numbering systems well known in the art, such as Kabat, Chothia, IMGT, etc., IMGT is used in this application. The variable region of heavy chain (VH) and variable region of light chain (VL) regions are responsible for antigen recognition, especially complementarity-determining regions (CDR), which are usually specific for different epitopes of antigens. The constant region is primarily responsible for effector functions.
The term “antigen-binding fragment” refers to an antigen-binding fragment of an antibody, that is, an antibody fragment that retains the ability to bind specifically to an antigen, such as a fragment that retains one or more CDR regions, including but not limited to Fab fragments, FV fragments, double antibodies, linear antibodies, single chain variable fragment, nanoantibodies, multi-specific antibodies, etc.
The term “monoclonal antibody” refers to a population of antibodies that is essentially homogeneous, in the sense that the amino acid sequences of the antibody molecules contained in the population are identical, except for a small number of present mutations that may occur naturally. In contrast, polyclonal antibodies typically include many different antibodies with different amino acid sequences in their variable domains, particularly their complementarity determining regions. Monoclonal antibodies may be obtained by methods known to one skilled in the art. This application uses hybridoma technique to obtain monoclonal antibodies, which is one of the many methods known to obtain monoclonal antibodies.
The term “murine antibody” refers to an antibody consisting only of a rat or mouse immunoglobulin sequence.
The variable region of “chimeric antibody” is derived from non-human (e.g., murine) antibody sequences, and the remainder is derived from human antibody sequences.
The CDR region of “humanized antibody” is derived from non-human (e.g., murine) antibody sequences, and the remainder is derived from human antibody sequences. The term “bispecific antibody” is an antibody that has two specific antigen-binding sites
of the same or different antigens.
The term “BiTE” is a bispecific antibody, full name “bispecific T cell engager”, formed by connecting two single chain variable fragments with antigen specificity through a linker. One scFv targets tumor-associated antigens and the other scFv targets CD3 on T cells. The single chain variable fragment (scFv) is formed by connecting the variable region of heavy chain and the variable region of light chain through a linker. Linker is an amino acid sequence used to link different protein fragments. There have been many studies on the design and selection of linker. In BiTE, the linker connects a variable region of heavy chain to a variable region of light chain to form scFv, and also concatenates two scFvs and allows them to rotate freely. It is generally composed of GGGGS repeats (S for serine, G for glycine). Glycine with small molecular weight and short side chain can increase the flexibility of side chain. The stronger hydrophilicity of serine may increase the hydrophilicity of the peptide chain. Optimizing the number of repeats not only helps the light and heavy chains in the scFv to form correct conformation, but also affects the distance between the two scFvs to promote the optimal interaction between T cells and target cells at the immune synapse (Zhouchong, etc. Research progress of bispecific single-chain antibody BiTE [J]. Chinese Journal of Biology, 2018″). KESGSVSSEQLAQFRSLD, GSTSGGGSGGGGGGSS (US20180326032), EGKSSGSGSESKST, GSTSGSGKPGSGEGSTKG (Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22) are commonly used to connect the variable region of heavy chain with the variable region of light chain in scFv. In a specific embodiment of the present application, the flexible linker connecting a heavy chain and a light chain to form scFv is composed of three GGGGS, and the flexible linker connecting two scFv is GGGGS. The linker can be selected and determined by one skilled in the art according to the teaching of the prior art through conventional experimental methods and finite tests from known linkers. The number of repeats of GGGGS in the two flexible linkers can be optimized, or other flexible linkers other than GGGGS can be used.
CD3 is a component of T-cell signaling. When BiTE binds to both T cells and tumor cells, T cells are activated, which promotes CD8+ T cells to directly secrete perforin and granzyme, and promotes CD4+T cells to secrete cytokines to recruit and activate killer T cells, thereby killing tumor cells. The CD3 antibody sequence can be selected from OKT-3, L2K, TR66, UCHT1, SP34, IORT3, Catumaxomab, Blinatumomab, Solitomab, WBP3311 2.306.4 (Patent CN201880061333.1) and other known CD3 antibody sequence in the art. In a specific embodiment of the application, the CD3 antibody sequence of Solitomab, WBP3311_2.306.4 is used, and the person skilled in the art can select and determine from other known CD3 antibodies by conventional experimental methods and a limited number of tests, rather than being limited to a specific CD3 antibody sequence of the application.
The term “chimeric BiTE bispecific antibody” refers to murine CD26 scFv connected to humanized CD3 scFv by a linker.
The terms “administration”, “administering”, “treating”, and “treatment” refer to the contact of an exogenous agent, therapeutic agent, or diagnostic agent with the tissues, organs, cells, or biological fluids of a subject. “Treatment” refers to slowing or preventing the development of clinical symptoms of the disease, or to alleviating or improving at least one physical parameter, or to preventing the progression of the disease.
According to the CD26 cDNA sequence published in GenBank (GenBank accession number: NM_001935.3), the rCD26 expression vector was designed, the histidine tag was introduced at the 5′end of the codon, the whole gene was synthesized, connected to the pCHO1.0 plasmid, and expressed in CHO-S cells. The target CD26 protein was purified. The prepared recombinant CD26 protein was used to immunize female BALB/c mice according to the general immunization schedule. For specific immunization, please refer to the «Making and Using Antibodies: A Practical Handbook». Indirect ELISA was used to detect the serum titer of immunized mice, and the immunized mouse with the highest serum titer were selected for fusion experiments with spleen cells and myeloma cells.
Eyeballs of immunized mouse were removed and blood was collected. The mouse was sacrificed by cervical vertebra amputation and immersed in 75% (v/v) alcohol for 10 minutes. The spleen was removed in a sterile operating table, placed in a cell screen, fully ground the cells, passed through the screen, centrifuged and washed with sterile 1640 medium (purchased from Gibco Company) for several times, and the cells were resuspended to make single cell suspension. And count and set aside.
A female BALB/c mouse aged 8-10 weeks was selected, and negative serum was obtained by enucleate the eyeball. The mouse was sacrificed by cervical vertebra amputation and immersed in 75% (v/v) alcohol for 10 minutes. The abdominal skin was aseptically uncovered to expose the peritoneum, and approximately 10 mL of 1640HT medium (purchased from SIGMA) was injected into the abdominal cavity of mouse using a syringe, the abdomen was gently massaged and blown several times. The medium containing macrophages was aspirated and injected into 20% 1640HAT medium for later use.
A female BALB/c mouse aged 2 to 3 weeks was sacrificed by cervical vertebra amputation and immersed in 75% (v/v) alcohol for 10 minutes. Thymocytes were aseptically removed, placed in a cell screen, ground, passed through the screen, and thymocytes were obtained and placed in 20% 1640HAT medium containing macrophages as described above for use.
Mouse myeloma cell line SP2/0 in logarithmic growth phase was selected, collected and counted. About 108 of the above spleen cells and 2×107 of the above SP2/0 cell lines were added to the fusion tube and mixed. After centrifugation at 1000 rpm for 10 minutes, the supernatant was discarded, and the fusion tube was placed on the palm and gently rubbed back and forth to loosen the precipitate. Add 1 mL of preheated PEG1450 (polyethylene glycol 1450, purchased from SIGMA) slowly and then quickly within 60 seconds, terminate by adding 30 mL of 1640HT medium, centrifuge at 1000 rpm for 10 minutes, remove the supernatant, gently rub to loosen the precipitate, and add to the 20% 1640HAT medium obtained in step 2.
The above HAT medium was mixed thoroughly, and 200 μL/well was sub-packed into a 96-well cell culture plate and incubated in a cell incubator at 37° C. with 5% CO2. One week later, 10% 1640HT medium was used to replace 20% 1640HAT medium, and the supernatant was taken after 3 days for assay.
(1) Preparation of Detection Plate: The Recombinant CD26 Protein was Diluted to 1 μg/Ml with CB coating solution, coated into 96-well ELISA plate, 100 μl/well, coated at 2-8° C. overnight, washed once and dry; The cells were blocked with 2% BSA in PBST buffer (200 μl/well) and then blocked at 37° C. for 2 hours. Dry and set aside.
(2). Screening of Positive Clones: The Cell Culture Supernatant was Added to the Test Plate, 100 μl/well, incubated at 37° C. for 30 minutes, washed and dried. Then 100 μl/well diluted HRP-labeled sheep anti-mouse IgG was added, incubated at 37° C. for 30 minutes, washed and dried, and 100 μl/well TMB chromogenic solution was added, incubated at 37° C. in the dark for 15 minutes. The reaction was terminated by adding 50 μl of 2M H2SO4 to each well, and the values were read at OD450. The positive wells was determined as follows: OD450 value/negative control value ≥2.1. Positive clones were selected for cell cloning screening. After three to four rounds of cloning and screening, the positive rate of monoclonal cell line was 100%, which was defined as stable cell line.
In the 786-0 cell reaction system, 786-0 cells were labeled with green fluorescent signal by fluorescent dye Calcein-AM. The 786-0 cells were seeded into U-type 96-well cell culture plate at a cell concentration of 6×105 cells/ml, 50 μl per well, and each group was set up with 3 parallel holes. Add 50 μl CD26 antibody at a final concentration of 100 ng/ml to the sample wells, add 50 μl medium control to the blank control wells, add 50 μl Triton X-100 at a final concentration of 1% to the positive control wells, and incubate at 37° C. for 30 min. PBMC cells (peripheral blood mononuclear cells) at a concentration of 6×106 cells/ml were added at E/T ratio of 10:1. The reaction system was incubated at 37° C. in carbon dioxide for 5 h. After the reaction, centrifuged and placed the supernatant in a new 96-well plate. After centrifugation again, 80 μl of the supernatant was placed in a black 96-well plate. The detection was performed with a microplate reader at 470 nm excitation wavelength and 515 nm emission wavelength. The formula of cell lysis rate=(Vsample−Vvehicle control)/(VTritonX-100−Vvehicle control)×100%. (Vsample is the mean value of the fluorescence signal reading measured at excitation and emission wavelengths in the drug treatment group, V vehicle control is the mean value of the fluorescence signal reading measured at excitation and emission wavelengths in the blank control group, VTritonX-100 is the mean value of fluorescence signal reading measured at excitation wavelength and emission wavelength in positive control group.)
The results showed that at the concentration of 100 ng/ml, No. 62 mouse antibody mediated lysis rate of 786-0 by PBMC was 53.1% after 5 h, which was much higher than that of other murine monoclonal antibodies, such as NO. 72, 160, 51, 212 mouse antibody, and was close to the humanized antibody YS110.
786-0 is a human tumor cell. Theoretically, the successful humanized antibody has a much stronger affinity with human cells than the parental murine antibody, and will also have a stronger lysis of the tumor cell. The killing effect of No. 62 mouse antibody in this application on human tumor cells has been equivalent to that of the humanized antibody YS110 which has completed the clinical phase II, proving the advantages of No. 62 mouse antibody. It is reasonable for one skilled in the art to expect that the humanized antibody derived from No. 62 mouse antibody will be superior to No. 62 mouse antibody itself and to YS110 in killing human tumor cells, which has also been verified in Example 11.
Step 1. Total RNA Extraction from CD26 Hybridoma Cells
The hybridoma cell lines were passaged into T75 culture flasks and cultured until the cells fusion reached about 90%. Cells were collected. Total RNA was extracted from the monoclonal hybridoma cell lines using an RNA extraction kit (purchased from Roche). Then, the total RNA was used as a template to amplify the first strand of cDNA by cDNA reverse transcription kit (purchased from Thermo), and the reaction products were stored at −20° C., or −70° C. for long-term storage.
The first strand cDNA of hybridoma cells was used as template, and 1 μl of cDNA, 5 μl of 10×PCR buffer, 1 μl of upstream and 1 μl of downstream primers (25 pmol), dNTP 1 μl, 1 μl of 25 mmol/L MgCl2, and 39 μl of H2O were added respectively to form the 50 μl reaction system. After initial denaturation at 95° C. for 10 min, 1 μl of Taq enzyme was added and entered into a temperature cycle for PCR amplification. The reaction condition was 94° C. Denaturation was performed at 58° C. for 1 min. There were 30 cycles of annealing for 1 min and extension at 72° C. for 1.5 min, followed by kept at 72° C. for 10 min. 5 μl of the PCR product was subjected to 1.2% agarose gel electrophoresis.
According to the instruction of pGM-T Fast ligation kit (Beijing Tiagen Biochemical Technology Co., Limited, VT207-02), the variable region genes of heavy and light chain were ligated with pGM-T vector, respectively, transformed into Escherichia coli Top10 competent cells, screened by Blue-White Screening and cultured at 37° C. for 12-16 hours.
The resulting white colonies were inoculated in 1-5 mL LB medium containing ampicillin at a final concentration of 100 μl and shaken at 37° C. for 3-4h. PCR was used to screen for clones with correct sequence insertion. The positive clones were screened by PCR and sequenced. The amino acid sequences of variable region of heavy chain and light chain and CDR region of the antibody were obtained by IMGT comparative analysis of the computer network gene library.
NO. 62 mouse antibodies included HCDR1 shown as SEQ ID NO: 1, HCDR2 shown as SEQ ID NO: 2, and HCDR3 shown as SEQ ID No: 3, and LCDR1 shown as SEQ ID NO: 4, LCDR2 composed of Arg Met Ser, where Arg represents arginine, Met represents methionine, and Ser represents serine, and LCDR3 shown as SEQ ID NO:5. Its variable region of heavy chain is shown as SEQ ID NO: 31, and its variable region of light chain is shown as SEQ ID NO:32.
Bispecific antibodies targeting both CD26 and CD3 were designed based on the heavy and light chain sequences of NO. 72, 160, 62, 51 murine monoclonal antibodies obtained from Example 1 and sequenced by the methods described in Example 2:
The variable region of light chain targeting CD26 and variable region of heavy chain targeting CD26 were connected to form the CD26 scFv regions by a short peptide (e.g. SEQ ID NO: 12). The sequences of scFv regions derived from NO. 72, 160, 62, 51 mouse antibody were shown as SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, respectively.
The variable region of heavy chain targeting CD3 (e.g., SEQ ID NO:10) and variable region of light chain targeting CD3 (e.g., SEQ ID NO:11) were connected to form the CD3 scFv region by a short peptide (e.g. SEQ ID NO:12). Four CD26 scFv regions are respectively fused to the CD3 scFv regions with a short peptide (e.g. SEQ ID NO:13) to form four BiTEs. The four BiTEs derived from NO. 72, 160, 62, 51 mouse antibody were 17G105, 17G108, 17G61, 17G131, shown as SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, respectively.
The mouse (Musmusculus) IgG kappa signal peptide (e.g., SEQ ID NO:24) was added upstream of the CD26 scFv amino acid sequence (SEQ ID NO:16-19). The optimized CD26 scFv gene sequence (SEQ ID NO: 25-28) was obtained according to the codon preference of mammalian cells CHO. And the AvrII restriction site and kozak sequence were introduced upstream. The scFv gene sequence of Solitomab anti CD3 was also optimized (SEQ ID NO:29). The linker gene (such as SEQ ID NO:30) was added upstream of the anti CD3 scFv gene, and the stop codon and BstZ17I restriction site were added downstream of the anti CD3 scFv gene. The AvrII restriction site, kozak sequence, optimized signal peptide, anti CD26 scFv, linker, anti CD3 scFv, stop codon and BstZ17I restriction site sequence were fused together to form four kinds of chimeric BiTE genes, which were synthesized and constructed into pUC57 plasmid. Several long-term preservation plasmids were formed, named pUC57-17G105, pUC57-17G108, pUC57-17G61 and pUC57-17G131, respectively.
The backbone vector pCHO 1.0 was purchased from Thermo Fisher. “Expression box 1” was removed by SfiI digestion and self-cycled with T4 ligase to form the pZHK2.0 vector. The target BiTE genes were amplified and the PCR products were purified after 1% agarose electrophoresis. The purified PCR products and the pZHK 2.0 vector were digested by AvrII and BstZ17I.
T4 ligase ligated the double-digested PCR product into the pZHK 2.0 vector and transformed into Top10 competent cells, which were plated on LB plates containing kanamycin resistance and cultured overnight at 37° C. The next day, positive clones were screened and sequenced, which were completely consistent with the expected sequence.
Mammalian CHO cell line stably and highly expressing the CD26-CD3 chimeric BiTE antibody was seeded in Dynamis medium (A2617501, Thermo Fisher), and fed batch culture was performed at 37° C., 8% CO2, and 130 rpm. The supernatant of the culture medium was centrifuged at 12000 rpm for 15 min at low-temperature. And then the supernatant was filtered through a 0.45 μm filter membrane, followed by chromatography purification. The size of the target product obtained was about 55 kDa, which was the correct size.
The affinity of the chimeric antibody to the antigen protein CD26 was detected by Fortebio Intermolecular Interaction Octet QKe instrument. First, the amino-coupled biosensor AR2G was activated with EDC/s-NHS, and the activated biosensor AR2G was solidified in CD26 protein solution diluted with 10 mM sodium acetate (pH 5.0) solution. The biosensor solidified with CD26 was quenched in 1M ethanolamine (pH8.5) solution. Then equilibrated in 1× kinetic solution. After equilibration, it was bound to a humanized bispecific antibody solution. It was dissociated in 1× kinetic solution. Fortebio Data Analysis 8.0 software was used to analyze the data and calculate the affinity constant (KD), association rate constant (kon) and dissociation rate constant (kdis). The Response value represents the number of antibody molecules that can bind to the antigen. Kon indicates the binding speed of the antibody to the corresponding receptor. Kdis represents the dissociation speed of antibody from corresponding receptor. NA indicates no valid data.
According to the above data, the chimeric antibody can bind CD26 and dissociate from CD26. 17G61 (anti-CD26-CD3 chimeric bispecific antibody derived from No. 62 mouse antibody) had the highest response value with CD26 protein, indicating that more 17G61 molecules bound to CD26 protein at the same concentration. When 17G105 (anti-CD26-CD3 chimeric bispecific antibody derived from No. 72 mouse antibody) and 17G131 (anti-CD26-CD3 chimeric bispecific antibody derived from No. 51 mouse antibody) bound to CD26 protein, the response values were significantly lower than those of 17G61. The KD value of 17G61 with CD26 is smaller, so 17G61 has a stronger affinity with CD26 protein.
The affinity of chimeric antibodies to antigen protein CD3 was determined by Fortebio Intermolecular Interaction Octet QKe instrument. First, the amino-coupled biosensor AR2G was activated with EDC/s-NHS, and the activated biosensor AR2G was solidified in CD3 protein solution diluted with 10 mM sodium acetate (pH 5.0) solution. The biosensor solidified with CD3 was quenched in 1M ethanolamine (pH8.5) solution. Then equilibrated in 1× kinetic solution. After equilibration, it was bound to bispecific antibody solution. They were dissociated in 1×kinetic solution. Fortebio Data Analysis 8.0 software was used to analyze the data and calculate the affinity constant value (KD).
According to the above data, 17G61 had the highest response value with CD3, which represented that more 17G61 molecules combined with CD3 protein at the same concentration. Binding of 17G105 to CD3 protein resulted in a lower response value than 17G61. These results indicate that 17G61 can bind CD3 and dissociate from CD3 protein.
NO. 62 and 212 murine antibody in Example 1, which have high CD26 affinity and strong killing activity against CD26 high expressing tumor cells, were humanized. Humanization analysis was performed on the variable regions of heavy chain (shown as SEQ ID NO: 31) and the variable regions of light chain (shown as SEQ ID NO: 32) of NO. 62 mouse antibody, as well as the variable regions of heavy chain (shown as SEQ ID NO: 33) and the variable regions of light chain (shown as SEQ ID NO: 34) of NO. 212 mouse antibody. After computer calculating and predicting, a number of humanized heavy and light chain combinations were obtained from the above two antibodies. Four different heavy and light chain amino acid sequences were obtained after humanization of NO. 62 mouse antibody: 18G272-VL (SEQ ID NO: 7), 18G272-VH (SEQ ID NO: 6), 19G292-VL (SEQ ID No: 35), 19G292-VH (SEQ ID NO: 36), 19G293-VL (SEQ ID NO: 37), 19G293-VH (SEQ ID NO: 38), 19G294-VL (SEQ ID NO: 9), 19G294-VH (SEQ ID NO: 8).
Four different heavy and light chain amino acid sequences were obtained after humanization of NO. 212 mouse antibody: 18G278-VL (SEQ ID NO: 39), 18G278-VH (SEQ ID NO: 40), 19G295-VL (SEQ ID NO: 41), 19G295-VH (SEQ ID NO: 42), 19G296-VL (SEQ ID NO: 43), 19G296-VH (SEQ ID NO: 44), 19G297-VL (SEQ ID NO: 45), 19G297-VH (SEQ ID NO: 46).
The humanized YS110 antibody was used as a positive control, and its heavy and light chain sequences are as follows: YS110-VL (SEQ ID NO: 47), YS110-VH (SEQ ID NO: 48).
The heavy and light chain of each humanized CD26 antibody (18G272, 19G292, 19G293, 19G294, 18G278, 19G295, 19G296, 19G297) were linked by a short peptide (SEQ ID NO: 12) respectively, and another short peptide (SEQ ID NO: 13) was added downstream. The optimized CD26 scFv gene sequence (SEQ ID NO: 49-56) was obtained after optimization, the AvrII restriction site and kozak sequence were introduced upstream, the CD3 scFv gene (SEQ ID NO: 29), stop codon, and BstZ17I restriction site were added downstream. The humanized CD26-CD3 BiTE gene sequence was obtained, synthesized and constructed into pUC57 plasmid. They were named pUC57-18G272, pUC57-19G292, pUC57-19G293, pUC57-19G294, pUC57-18G278, pUC57-19G295, pUC57-19G296, pUC57-19G297.
At the same time, the heavy and light chain of YS110 antibody were also linked by a short peptide (SEQ ID NO: 12), another short peptide (SEQ ID NO: 13) was added downstream, then the AvrII restriction site and kozak sequence were introduced upstream. The CD3 scFv gene (SEQ ID NO: 29), stop codon, and BstZ17I restriction site were added downstream. The humanized CD26-CD3 BiTE gene sequence was obtained, synthesized and constructed into pUC57 plasmid, named pUC-17G29.
In addition, The heavy and light chain of CD3 antibody from WBP3311_2.306.4 were linked by short peptide (SEQ ID NO: 12) to form a scFv (SEQ ID NO: 57). The heavy and light chain of 19G294 were linked by short peptide (SEQ ID NO: 12), and another short peptide (SEQ ID NO: 13) was added downstream, then conjugated with the scFv (SEQ ID NO: 57).
Optimization was performed according to the CHO cell codon preference. The AvrII restriction site and kozak sequence were introduced upstream, and the stop codon and BstZ17I restriction site were added downstream. It was directly synthesized into pUC57 plasmid and named puc57-21G587.
The target gene was amplified and the PCR product was recovered by 1% agarose electrophoresis. The recovered PCR product and pZHK2.0 vector were digested by AvrII and BstZ17I. T4 ligase ligated the double digestion product into pZHK2.0 vector and transformed into Top10 competent cells. The cells were plated on LB plates containing kanamycin resistance and incubated overnight at 37° C. The next day, the positive clones were screened and sequenced. The sequence was completely consistent with the expected sequence, and the humanized CD26-CD3 BiTE expression plasmid was obtained.
The mammalian cell line with high expression of CD26-CD3 humanized BiTE was cultured in Dynamis medium, and fed batch culture was performed at 37° C., 8% CO2 and 130 rpm. The supernatant of the culture medium was centrifuged at 12000 rpm for 15 min to remove the cell pellet at low-temperature. And then the supernatant was filtered through a 0.45 μm filter membrane, followed by chromatography purification. The target products were all about 55 kDa.
The purified sample was diluted into different concentration gradients, 2 μl of the diluted sample was placed on the NC membrane, and the positive control sample was 17G29. After complete absorption, the membrane was blocked with 5% skim milk solution, shaked at room temperature for 1 hour. The membrane was washed 3 times with TBST for 5 min each time.
CD3-HRP protein was added to 2.5% skim milk solution at 1:1000 (50 μl CD3-HRP was added to 50 ml of 2.5% skim milk solution), shaked at room temperature for 1 hour, and washed three times with TBST for 5 min each time. CD26-HRP protein was added to 2.5% skim milk solution at 1:1000 (50 μl CD26-HRP was added to 50 ml of 2.5% skim milk solution), shaked at room temperature for 1 h, and washed three times with TBST for 5 min each time. The TBST solution on the film was blotted, and the color development solution (SuperSignal West Pico Chemiluminescent Substrate) was added. After 1 min of color development, the color development solution was blotted and exposed in the gel imaging system. The continuous exposure program was selected and set to 15 s/sheet. Twelve consecutive exposures were made. Dot blot analysis of 18G272 (humanized antibody derived from NO. 62 mouse antibody and 18G278 (humanized antibody derived from NO. 212 mouse antibody) showed that the binding activity of 18G272 to CD26 and CD3 at different concentrations was close to that of the positive control 17G29 (anti-CD26-CD3 humanized bispecific antibody derived from YS110). It was significantly higher than that of 18G278 (
The target cells were cultured in T75 cell culture flask. When the cell fusion was more than 80%, the cells were digested by trypsin and collected, washed once with PBS, and counted by a hemocytometer. The cells were divided into 5×105 cells each. Anti-CD26 monoclonal antibody was used as the primary antibody, and incubated with the target cells at room temperature for about 40 min. After incubation and centrifugation, the supernatant was discarded, and the cell precipitate was resuspended in PBS. The supernatant was discarded after centrifugation again, and the cell precipitate was collected. Then Alexa Fluor 488 mouse anti-human IgG1 was used as the secondary antibody. The cells were resuspended and incubated at room temperature in a dark place for about 30 min. After incubation, the cells were washed twice with PBS, the supernatant was discarded after centrifugation, and the cell precipitates were collected. The cells were resuspended in 200 μl of PBS, and the positive rate of CD26 was analyzed by flow cytometer within 1h.
Identified by flow cytometer, CD26 positive cell lines were 786-0, OS-RC-2, A498, NCI-H226, NCI-H2052, NCI-H596, HCC827, Huh-7 and PC-3, and CD26 negative cell lines were G401 and A-375. Their positive rates are shown in Table 4. The CD26 positive cell line was consistent with the reference (Chinese Patent application number CN200680034937.4, CD26/DPP4-a potential biomarker and target for cancer therapy, Pharmacology & Therapeutics (2019), 198:135-159).
The fluorescein FITC was weighed and dissolved in DMSO solution to a final concentration of 1 mg/ml. 500 μl of 18G272 antibody was added to a final concentration of 1 mg/ml, and 1/10 volume of 1M Na2CO3 solution was added, and 15 μl of FITC solution was added. Mix well. The mixture was incubated at room temperature in the dark for 3.5h. Then, the mixture was centrifuged with an ultrafiltration tube with an interception diameter of 10 KDa to completely remove FITC that was not bound to 18G272, and the solution in the tube was collected.
When the cell fusion was about 80%, the cells cultured in T75 cell culture flask were digested with trypsin and collected. The cells were resuspended in an appropriate amount of PBS, and counted with a hemacytometer, and divided into 5×105 cells each. The cell precipitates were collected after centrifugation. The target cells were resuspended with FITC-labeled 18G272 solution, placed on a homogenizer, and incubated at room temperature in the dark for 30 min. After incubation, the cells were resuspended in PBS, the supernatant was discarded after centrifugation, and the operation was repeated once. Cell precipitates were resuspended in 500 μl of PBS solution. Negative control: In another sample, no antibody sample was added in the labeling process, the remaining steps were performed in parallel with the above process, and the resulting solution was the FITC-labeled negative control. A BD Accuri C6 flow cytometer was used to analyze the positive rate of FITC labeling.
The results showed that the humanized bispecific antibody 18G272 could bind to CD26 positive cells, such as 786-0 cells, NCI-H226 cells, PC-3 cells and HCC 827 cells, but not to CD26 negative A375 cells. Its binding to the CD26 target is specific.
The same method was used to detect the binding specificity of the humanized bispecific antibody 19G294 to the target cell antigen. 19G294 could bind to CD26 positive cells, such as 786-0 cells, NCI-H226 cells, PC-3 cells, OS-RC-2 cells, but not to CD26 negative G401 cells. The humanized bispecific antibody 19G294 could bind to CD26 positive cells but not to CD26 negative cells. Its binding to the CD26 target is specific.
Representative figures of binding specificity of antibody to target cell antigens determined by flow cytometry were shown in
Other humanized bispecific antibodies of the present invention also bind to CD26 positive cells, but not to CD26 negative cells, and have specific binding to cells with CD26 antigen.
1, Affinity Study with CD26 Protein
CD26 protein was biotinylated with EZ-link NHS-PEG12-Biotin. Biotinylated CD26 protein was immobilized to SA biosensor. Equilibrated in 1×Kinetics solution. The CD26 immobilized to SA biosensor was combined with the antibody solution to be detected. The concentration gradients of the antibody solution were 31.3 nM, 62.5 nM, 125 nM, 250 nM, and 500 nM. The biosensor was dissociated in 1×Kinetics solution. Fortebio Data Analysis 8.0 software was used for data analysis and affinity constant values were calculated.
According to the above data, when the anti-CD3 segment was the same, the affinity constant of the humanized antibodies (19G292, 19G293, 19G294) derived from NO. 62 mouse antibody was lower than that of the humanized antibodies derived from NO. 212 mouse antibody or YS110, indicating that the former had a higher affinity to CD26. In addition, 19G294 and 21G587 had the same anti-CD26 segment but different anti-CD3 segment, and both had the same level of affinity with CD26 protein.
CD3 protein was biotinylated with EZ-link NHS-PEG12-Biotin. Biotinylated CD3 protein was immobilized to SA biosensor. Equilibrated in 1×Kinetics solution. The CD3 immobilized to SA biosensor was combined with the humanized bispecific antibody solution to be detected. The concentration gradient of humanized bispecific antibody was 200 nM, 400 nM, 800 nM, and 1600 nM. The biosensor was dissociated in 1×Kinetics solution. Fortebio Data Analysis 8.0 software was used for data analysis and affinity constant values were calculated.
In this experiment, the affinity constant of 19G294 to CD3 protein was 0.751 nM. After binding to CD3, 19G296 and 19G297 dissociated rapidly. With the same anti-CD3 segment, the affinity of humanized bispecific antibodies 19G294, 19G292 and 19G293 derived from No. 62 mouse antibody to CD3 protein was significantly higher than that of humanized bispecific antibody 17G29 derived from YS110. In addition, 19G294 and 21G587 have the same anti-CD26 segment but different anti-CD3 segment, and both have the same level of affinity with CD3 protein.
Combined with the affinity to CD26 protein (Table 7) and CD3 protein (Table 8) of the above antibodies, the humanized antibodies derived from No. 62 mouse antibodies (19G292, 19G293, 19G294) showed better CD26 protein binding ability than the humanized antibodies derived from No. 212 mouse antibodies (19G296, 19G297). The humanized antibodies (19G292, 19G293, 19G294) derived from No. 62 mouse antibodies showed better CD3 binding and dissociation ability than those derived from No. 212 mouse antibodies (19G296, 19G297) and 17G29 humanized antibodies derived from YS110. 19G294 is a more advantageous combination of bispecific antibody molecules.
The 19G294 protein was biotinylated with EZ-link NHS-PEG12-Biotin. The biotinylated 19G294 protein was immobilized to SA biosensor. Equilibrated in 1×Kinetics solution; The antibody (19G294 protein) immobilized to SA biosensor was first combined with 400 nM CD26 protein solution, and then placed in 400 nM CD26 solution containing 400 nM CD3 protein. Or, the antibody (19G294 protein) immobilized to SA biosensor was first combined with 400 nM CD3 protein solution, and then placed in 400 nM CD3 solution containing 400 nM CD26 protein. Fortebio Data Analysis 8.0 software was used to observe the binding.
As shown in
In the 786-0 cell reaction system, 786-0 cells were labeled with green fluorescent signal by fluorescent dye Calcein-AM. The 786-0 cells were seeded into U-type 96-well cell culture plate at a cell concentration of 6×105 cells/ml, 50 μl per well. 50 μl of humanized bispecific antibody with final concentrations of 100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml, 0.01 ng/ml and 0.001 ng/ml were added to the corresponding sample reaction wells. 50 μl of culture medium was added to the blank control wells. Add 50 μl of 1% TritonX-100 to the positive control well. Then add 50 μl of PBMC cells with a concentration of 6×106 cells/ml at the E/T ratio of 10:1, and incubate the reaction system at 37° C. in carbon dioxide for 5 h. After the reaction and centrifugation, the supernatant was placed in a new 96-well plate, and after centrifugation again, 80 μl of the supernatant was placed in a black 96-well plate for detection at 470 nm excitation wavelength and 515 nm emission wavelength using a microplate reader.
In OS-RC-2 cell reaction system, OS-RC-2 cells were labeled with green fluorescent signal by fluorescent dye Calcein-AM. OS-RC-2 cells were seeded into U-type 96-well cell culture plate at a cell concentration of 6×105 cells/ml, 50 μl per well. 50 μl of humanized bispecific antibody with final concentrations of 100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml, 0.01 ng/ml and 0.001 ng/ml were added to the corresponding sample reaction wells. 50 μl of culture medium was added to the blank control wells. Add 50 μl of 1% TritonX-100 to the positive control well. Then add 50 μl of PBMC cells with a concentration of 9×106 cells/ml at the E/T ratio of 15:1, and incubate the reaction system at 37° C. in carbon dioxide for 5 h. After the reaction and centrifugation, the supernatant was placed in a new 96-well plate, and after centrifugation again, 80 μl of the supernatant was placed in a black 96-well plate for detection at 470 nm excitation wavelength and 515 nm emission wavelength using a microplate reader.
In the NCI-H226 cell reaction system, NCI-H226 cells were labeled with green fluorescent signal by fluorescent dye Calcein-AM, and NCI-H226 cells were seeded into U-type 96-well cell culture plate at a cell concentration of 6×105 cells/ml, 50 μl per well. 50 μl of humanized bispecific antibody with final concentrations of 100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml, 0.01 ng/ml and 0.001 ng/ml were added to the corresponding sample reaction wells. 50 μl of culture medium was added to the blank control wells. Add 50 μl of 1% TritonX-100 to the positive control well. Then add 50 μl of PBMC cells with a concentration of 9×106 cells/ml at the E/T ratio of 15:1, and incubate the reaction system at 37° C. in carbon dioxide for 5 h. After the reaction and centrifugation, the supernatant was placed in a new 96-well plate, and after centrifugation again, 80 μl of the supernatant was placed in a black 96-well plate for detection at 470 nm excitation wavelength and 515 nm emission wavelength using a microplate reader.
In the NCI-H2052 cell reaction system, NCI-H2052 cells were labeled with green fluorescent signal by fluorescent dye Calcein-AM, and NCI-H2052 cells were seeded into U-type 96-well cell culture plate at a cell concentration of 6×105 cells/ml, 50 μl per well. 50 μl of humanized bispecific antibody with final concentrations of 100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml, 0.01 ng/ml and 0.001 ng/ml were added to the corresponding sample reaction wells. 50 μl of culture medium was added to the blank control wells. Add 50 μl of 1% TritonX-100 to the positive control well. Then add 50 μl of PBMC cells with a concentration of 9×106 cells/ml at the E/T ratio of 15:1, and incubate the reaction system at 37° C. in carbon dioxide for 5 h. After the reaction and centrifugation, the supernatant was placed in a new 96-well plate. After centrifugation again, 80 μl of the supernatant was placed in a black 96-well enzyme plate and detected with a microplate reader at 470 nm excitation wavelength and 515 nm emission wavelength.
In the PC-3 cell reaction system, PC-3 cells were labeled with green fluorescent signal by fluorescent dye Calcein-AM. PC-3 cells were seeded into U-type 96-well cell culture plate at a cell concentration of 6×105 cells/ml, 50 μl per well. 50 μl of humanized bispecific antibody with final concentrations of 100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml, 0.01 ng/ml and 0.001 ng/ml were added to the corresponding sample reaction wells. 50 μl of culture medium was added to the blank control wells. Add 50 μl of 1% TritonX-100 to the positive control well. Then add 50 μl of PBMC cells with a concentration of 9×106 cells/ml at the E/T ratio of 15:1, and incubate the reaction system at 37° C. in carbon dioxide for 5 h. After the reaction and centrifugation, the supernatant was placed in a new 96-well plate, and after centrifugation again, 80 μl of the supernatant was placed in a black 96-well plate for detection at 470 nm excitation wavelength and 515 nm emission wavelength using a microplate reader.
In the HCC 827 cell reaction system, HCC 827 cells were labeled with green fluorescent signal by fluorescent dye Calcein-AM. HCC 827 cells were seeded into U-type 96-well cell culture plate at a cell concentration of 6×105 cells/ml, 50 μl per well. 50 μl of humanized bispecific antibody with final concentrations of 100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml, 0.01 ng/ml and 0.001 ng/ml were added to the corresponding sample reaction wells. 50 μl of culture medium was added to the blank control wells. Add 50 μl of 1% TritonX-100 to the positive control well. Then add 50 μl of PBMC cells with a concentration of 6×106 cells/ml at the E/T ratio of 10:1, and incubate the reaction system at 37° C. in carbon dioxide for 5 h. After the reaction and centrifugation, the supernatant was placed in a new 96-well plate. After centrifugation again, 80 μl of the supernatant was placed in a black 96-well enzyme plate and detected with a microplate reader at 470 nm excitation wavelength and 515 nm emission wavelength.
Cell lysis rate=(Vsample−Vvehicle control)/(VTritonX-100−Vvehicle control)×100%. (Vsample is the mean value of the fluorescence signal reading of the drug treatment group, V vehicle control is the mean value of the fluorescence signal reading of the blank control group, and VTriton-100 is the mean value of the fluorescence signal reading of the positive control group.)
Cell lysis rate and sample concentration values were used to calculate the IC50 value of samples mediated PBMC cytotoxicity against target cells using GraphPad Prism 7.00 software.
Anti-CD26-CD3 humanized bispecific antibodies derived from No. 62 mouse antibody, such as 18G272, 19G292, 19G293 and 19G294, can mediate PBMC cytotoxicity against a variety of CD26 positive tumor cells, and had killing activity in vitro. Anti-CD26-CD3 humanized bispecific antibodies such as 19G295, 19G296 and 19G297 derived from No. 212 mouse antibody did not mediate PBMC cytotoxicity against CD26 positive tumor cells.
On the one hand, the mouse antibody screened in this application have better killing activity against CD26 positive tumor cells. The anti-CD26-CD3 humanized antibody derived from No. 62 mouse antibody had better killing effect on a variety of CD26 positive tumors than 17G29 (the anti-CD26-CD3 humanized antibody derived from No. 62 mouse antibody) and the anti-CD26-CD3 humanized antibody derived from No. 212 mouse antibody. On the other hand, the BiTE (18G272, 19G292, 19G293, 19G294, 17G29) presented in this study showed better killing effects on a variety of CD26 positive tumor cells than the full-length IgG antibody YS110.
PBMC cells+bispecific antibody system: PBMC cells were collected, counted, resuspended to 1×106 cells/ml, 200 μl per well was added to 12-well cell culture plates, antibody was diluted to a final concentration of 1 ng/ml, and co-incubated for 24 hours. PBMC cell+bispecific antibody+OS-RC-2 cell system: PBMC cells were collected, counted, resuspended to 6×105 cells/ml, 100 μl/well was added to 12-well cell culture plates, antibody was diluted to a final concentration of 1 ng/ml, OS-RC-2 cells were diluted to 9×106 cells/ml, 100 μl per well was added to the plates, co-incubated for 24h.
After incubation and centrifugation, the cell precipitate was collected, resuspended in PBS, added anti-CD25-APC antibody (purchased from Miltenyl Company) and anti-CD69-PE antibody (purchased from Miltenyl Company), incubated at 4° C. for 10 min, and washed twice with PBS. After resuspension with PBS, the samples were analyzed by BD ACCURI C6 flow cytometer.
In the “PBMC+bispecific antibody” system, bispecific antibody did not induce the upregulation of CD25 and CD69 in PBMC. In the system of “PBMC+bispecific antibody+OS-RC-2”, bispecific antibody induced the upregulation of CD25 and CD69 in PBMC and promoted the activation of PBMC. In the system of “PBMC+YS110+OS-RC-2”, YS110 did not induce the upregulation of CD25 and CD69 expression in PBMC. See Table 10.
The same phenomenon was also observed in other CD26-positive tumor cells such as 786-0. In the “PBMC+bispecific antibody” system, bispecific antibody did not induce the upregulation of CD25 and CD69 expression in PBMC cells. The expression of CD25 and CD69 in PBMC cells was induced by “PBMC cells+bispecific antibody+other CD26 positive tumor cells (786-0, etc.)”.
CD25 and CD69 are important markers of T cell activation. The above results demonstrated that the full-length IgG antibody YS110 does not effectively induce T cell activation. CD26-CD3 bispecific antibody does not cause significant T cell activation before reaching the tumor. Bispecific antibody can cause T cells activation around the tumor after binding to tumor cells, and then kill the tumor cells, and improves the safety of the action.
OS-RC-2 cells were resuspended to 6×105 cells/ml, and 100 μl per well was added to 12-well plate. Single donor derived PBMC cells were collected and resuspended to 9×106 cells/ml, 100 μl was added to each well, and 100 μl of 19G294, 18G272 and 17G29 samples with concentrations of 0 ng/ml, 3 ng/ml were added respectively, and the final volume was 300 μl. The reaction system was incubated at 37° C. for 2 h, 4h, 6h, and 21h, and the culture supernatant was collected. After centrifugation, the supernatant was taken for ELISA detection. The levels of IFN-γ, TNF-α, L-4 and IL-10 were detected by ELISA kit.
ELISA kits were used to detect the cytokine content of each sample as shown in the table below.
* indicates that the reading value is too high to exceed the device detection limit.
In summary, in the process of humanized bispecific antibody-mediated killing effect of PBMC on target cells, the cytokines secreting of PBMC introduced by anti-CD26-CD3 humanized bispecific antibodies 19G294 and 18G272 derived from No. 62 mouse antibody were lower than those induced by anti-CD26-CD3 humanized bispecific antibodies 17G29 derived from YS110 at all time points with the same dosage. For this type of molecular structure targeting CD3 and activating T cells, low cytokine storm caused by cytokine secretion of T cells is associated with higher safety. (A FAD oncology analysis of CD3 bispecific constructs and first-in-human dose selection (2017), Regulatory Toxicology and Pharmacology, 90:144-152). The bispecific antibodies 19G294 and 18G272 of this application had the advantage of inducing lower cytokines secreting of T cells, which may be related to recognizing different CD26 epitopes with 17G29. Compared with other epitopes, the antigen epitope recognized by the proposed antibody is related to reducing the risk of cytokine storm and improving safety.
PBMC cells were labeled with green fluorescent signal by fluorescent dye Calcein-AM. 786-0 cells were seeded into U-type 96-well cell culture plate at a cell concentration of 6×105 cells/ml, 50 μl per well. 50 μl of humanized bispecific antibody with final concentrations of 100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml, 0.01 ng/ml and 0.001 ng/ml were added to the corresponding sample reaction wells. 50 μl of culture medium was added to the blank control wells. 50 μl TritonX-100 (final concentration 1%) was added to the positive control well. Then 50 μl PBMC cells with concentration of 9×106 cells/ml were added at the E/T ratio of 10:1. The reaction system was incubated at 37° C. in carbon dioxide for 5 h. After the reaction and centrifugation, the supernatant was placed in a new 96-well plate. After centrifugation again, 80 μl of the supernatant was placed in a black 96-well enzyme plate and detected using a microplate reader at 470 nm excitation light wavelength and 515 nm emission wavelength.
PBMC cells were labeled with green fluorescent signal by fluorescent dye Calcein-AM. OS-RC-2 cells were seeded into U-type 96-well cell culture plate at a cell concentration of 6×105 cells/ml, 50 μl per well. 50 μl of humanized bispecific antibody 18G272 with final concentrations of 100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml, 0.01 ng/ml and 0.001 ng/ml were added to the corresponding sample reaction wells. 50 μl of culture medium was added to the blank control wells. 50 μl TritonX-100 (final concentration of 1%) was added to the positive control well. Then 50 μl PBMC cells with concentration of 9×106 cells/ml were added at the E/T ratio of 15:1. The reaction system was incubated at 37° C. in carbon dioxide for 5 h. After the reaction and centrifugation, the supernatant was placed in a new 96-well plate. After centrifugation again, 80 μl of the supernatant was placed in a black 96-well enzyme plate and detected using a microplate reader at 470 nm excitation light wavelength and 515 nm emission wavelength.
Cell lysis rate=(Vsample−Vvehicle control)/(VTritonX-100−Vvehicle control)×100%. (Vsample is the mean value of the fluorescence signal reading of the drug treatment group, Vvehicle control is the mean value of the fluorescence signal reading of the solvent control group, and VTriton-100 is the mean value of the fluorescence signal reading of the positive control group.) The IC50 values of each sample mediated toxicity of PBMC to PBMC were calculated by GraphPad Prism 7.00 software.
In the process of PBMC killing 786-0 mediated by antibody 18G272, the ability of PBMC to kill PBMC cells was very low. The IC50 value of 18G272-mediated cytotoxicity of PBMC (4 #) against PBMC (4 #) cells was 99730000 pg/ml. PBMC (6 #) had no 18G272-mediated cytotoxic effect on PBMC (6 #) cells. The results are shown in
In the process of PBMC killing OS-RC-2 mediated by antibody 18G272, 18G272 did not mediate killing effect of PBMC to PBMC cells. PBMC (1 #) had no 18G272-mediated cytotoxic effect on PBMC (1 #) cells. PBMC (3 #) had no 18G272-mediated cytotoxic effect on PBMC (3 #) cells.
In the process of PBMC killing 786-0 mediated by antibody 19G294, the ability of PBMC to kill PBMC cells was very low. The IC50 value of 19G294-mediated cytotoxicity of PBMC (2 #) against PBMC (2 #) cells was 1915313 pg/ml. The IC50 value of 19G294-mediated cytotoxicity of PBMC (3 #) against PBMC (3 #) cells was 1947224 pg/ml.
In the process of PBMC killing OS-RC-2 mediated by antibody 19G294, the ability of PBMC to kill PBMC cells was very low. The IC50 value of 19G294-mediated cytotoxicity of PBMC (2 #) against PBMC (2 #) cells was 47920315 μg/ml. The IC50 value of 19G294-mediated cytotoxicity of PBMC (3 #) against PBMC (3 #) cells was 1406115138 pg/ml.
In summary, CD26-CD3 humanized bispecific antibody can accurately recognize PBMC. Although it has been reported that T cells express CD26, the bispecific antibodies 18G272 and 19G294 in this application do not mediate cytotoxicity of PBMC cells to PBMC cells obviously, which proved that it has good safety.
The target cells were cultured in T75 cell culture flask. When the cell fusion was more than 80%, the cells were digested by trypsin and collected, washed once with PBS, and counted by a hemocyte counter, and divided into 5×105 cells each. The target cells were incubated with PE-labeled anti-PD-L1 monoclonal antibody (purchased from Sino Biological Company) for about 40 min at room temperature in the dark. After incubation and centrifugation, the supernatant was discarded, the cell precipitate was resuspended in PBS, the supernatant was discarded after centrifugation again, the cell precipitate was collected, and resuspended in about 200 μl of PBS solution. The positive rate of PD-L1 was detected by flow cytometry within 1 hour.
By flow cytometry, the PD-L1 positive rate was 66.2% in 786-0 cell line and 29.6% in A498 cell line.
Human renal carcinoma 786-0-luc cells (the Luciference reporter gene was transfected into 786-0 cells) and human renal carcinoma A498-luc cells (the Luciference reporter gene was transfected into A498 cells) were seeded into white bottom opaque 96-well cell culture plates at a concentration of 3×105 cells/ml, 50 μl per well. 50 μl of antibody at final concentrations of 100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml, 0.01 ng/ml, and 0.001 ng/ml was added to the corresponding reaction wells, respectively. Then 50 μl PBMC cells with a concentration of 3×106 cells/ml were added at the E/T ratio of 10:1. The reaction system was incubated at 37° C. in carbon dioxide for 21 h. After the reaction, the reaction system was centrifuged, the supernatant was discarded, and 100 μl reporter gene detection reagent (purchased from Vazyme Company) was added. After shaking at 400 rpm for 5 min on a microplate oscillator, the assay was performed immediately with a microplate reader under Lum conditions. GraphPad Prism 7.00 software was used to make a 4-parameter curve according to the reduced level of reporter gene and the dose of antibody, and calculated the EC50 value. The results were shown in Table 16.
According to the above data, the EC50 value of 19G294 mediated PBMC cytotoxicity against two target cells was much lower than that of BAVENCIO. The expression rates of PD-L1 in 786-0 and A498 were 66.2% and 29.6%, respectively. 19G294 was significantly more active than BAVENCIO regardless of the level of PD-L1 expression.
Human renal carcinoma 786-0-luc cells (the Luciference reporter gene was transfected into 786-0 cells) and human renal carcinoma A498-Luc cells (the Luciference reporter gene was transfected into A498 cells) were seeded into white bottom opaque 96-well cell culture plates at a concentration of 3×105 cells/ml, 50 μl per well. 50 μl of antibody at final concentrations of 100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml, 0.01 ng/ml, and 0.001 ng/ml was added to the corresponding reaction wells, respectively. Then 50 μl PBMC cells with a concentration of 3×106 cells/ml were added at the E/T ratio of 10:1. The reaction system was incubated at 37° C. in carbon dioxide for 21 h. After the reaction, the reaction system was centrifuged, the supernatant was discarded, and 100 μl reporter gene detection reagent (purchased from Vazyme Company) was added. After shaking at 400 rpm for 5 min on a microplate oscillator, the assay was performed immediately with a microplate reader under Lum conditions. GraphPad Prism 7.00 software was used to make a 4-parameter curve according to the reduced level of reporter gene and the dose of antibody, and calculated the EC50 value. The results were shown in Table 17.
From the above data, it can be seen that when the anti-CD26 antibody fragment of the patent was combined with different anti-CD3 fragment, 19G294 and 21G587 had the same anti-CD26 fragment and different anti-CD3 fragments, and both had the same level of in vitro efficacy.
Methods: 5×106 cells/0.1 ml PC-3 cell suspension and 1×107 cells/0.1 ml PBMC cell suspension were fully mixed in a volume ratio of 1:1 and inoculated subcutaneously into NOD/SCID mice aged 5 to 7 weeks weighing 18 to 22 g, 0.2 mL per mouse. According to the body weight, the animals were randomly divided into three groups: Model group (PBS), 19G294 group (30 μg per mouse once daily), 18G272 group (30 μg per mouse once daily), with 6 animals in each group. Intravenous treatment with humanized bispecific antibodies (18G272 and 19G294) or the vehicle (PBS) started on the day of inoculation, continuously for 5 days. Two days after drug withdrawal, a second course of treatment was performed. The first treatment was 1h after inoculation, that is, the first courses were D1, D2, D3, D4, D5. The second courses were D8, D9, D10, D11 and D12. The frequency of treatment was once a day.
General clinical observation: General clinical observations were conducted at least once daily during the quarantine period and trial period, including the tumor growth and the effect of treatment on normal behavior of animals, including the death or near death, mental status, animal behaviour and other abnormal behaviour of tumor-bearing mice.
Body weight: Body weight was conducted twice or thrice a week.
Tumor volume: Tumor volume was conducted twice or thrice a week. The length and width of the tumor were measured by vernier caliper. Tumor volume: V=(length×width2)/2. Tumor Growth Inhibition value: TGI (%)=(1−T/C)×100%. It was generally accepted that T represented the relative tumor volume (the ratio of the tumor volume at the time of measurement to the tumor volume at the time of grouping) in the administration group and C represented the relative tumor volume (the ratio of the tumor volume at the time of measurement to the tumor volume at the time of grouping) in the model group. However, mice in this trial were divided into groups according to the body weight after inoculation. The TGI was calculated according to the T and C that represented the actual tumor volume of the treatment group and control group.
Tumor weight: Mice were euthanized at the end of the experiment, the tumor mass was stripped, rinsed with normal saline and blotted dry with filter paper, weighed and photographed. Tumor Growth Inhibition value: TGI (%)=(1−TTW/CTW)×100%, TTW represented average tumor weight of treatment group, CTW represented average tumor weight of control group.
Conclusion: 18G272 had a tendency to inhibit the tumor growth of mice bearing PC-3 (human prostate cancer) at the dose of 30 μg per mouse, and 19G294 can almost completely inhibit the tumor growth of mice bearing PC-3. These results were shown in
Methods: 8×106 cells/0.1 ml NCI-H596 cell suspension and 1.6×107 cells/0.1 ml PBMC cell suspension were fully mixed in a volume ratio of 1:1 and inoculated subcutaneously into NOD/SCID mice aged 5 to 7 weeks weighing 18 to 22 g, 0.2 mL per mouse. According to the body weight, the animals were randomly divided into two groups: Model group (PBS, 6 mice/group) and 18G272 group (30 μg per mouse once daily, 6 mice/group).
Administration route and administration frequency were the same as in Example 17.
General clinical observation, body weight, and tumor volume were conducted as in example 17.
Conclusion: 18G272 can significantly inhibit the tumor growth of mice bearing NCI-H596 (human lung adenosquamous carcinoma) at the dose of 30 μg per mouse. Mice in 18G272 group had an upward trend in body weight and were well tolerated during treatment at the dose of 30 μg per mouse. The results were shown in
Methods: 5×106 cells/0.1 ml NCI-H226 cell suspension and 1.5×107 cells/0.1 ml PBMC cell suspension were fully mixed in a volume ratio of 1:1 and inoculated subcutaneously into NOD/SCID mice aged 5 to 7 weeks weighing 18 to 22 g, 0.2 mL per mouse. The animals were randomly divided into two groups according to body weight: Model group (PBS, 5 mice/group) and 19G294 group (60 μg per mouse once daily, 5 mice/group). Administration route and administration frequency were the same as in Example 17.
General clinical observation, body weight, and tumor volume were conducted as in example 17.
Conclusion: 19G294 can significantly inhibit the tumor growth of mice bearing NCI-H226 human lung adenosquamous carcinoma (mesothelioma) at the dose of 60 μg per mouse, as shown in
a(%)
aindicates TGI calculated according to tumor weight on day 57 after cell inoculation.
Methods: 1×107 cells/0.1 ml A498 cell suspension and 1×107 cells/0.1 ml PBMC suspension were fully mixed in a volume ratio of 1:1 and inoculated subcutaneously into NOD/SCID mice aged 5 to 7 weeks weighing 18 to 22 g, 0.2 mL per mouse. According to the body weight, the animals were randomly divided into two groups: Model group (PBS) and 19G294 group (30 μg per mouse once daily), with 5 animals in each group.
Administration route and administration frequency were the same as in Example 17.
General clinical observation, body weight, tumor volume, and tumor weight were conducted as in example 17.
Conclusion: 19G294 can completely inhibit the tumor growth of mice bearing A498 human renal cell carcinoma at the dose of 30 μg per mouse, as shown in
Methods: 5×106 cells/0.1 ml OS-RC-2 cell suspension and 1×107 cells/0.1 ml PBMC cell suspension were fully mixed in a volume ratio of 1:1 and inoculated subcutaneously into NOD/SCID mice aged 5 to 7 weeks weighing 18 to 22 g, 0.2 mL per mouse. The animals were randomly divided into three groups according to the body weight: Model group (PBS), 17G29 group (15 μg per mouse once daily), 19G295 group (15 μg per mouse once daily). There were 8 animals in each group.
Administration route and administration frequency were the same as in example 17.
General clinical observation, body weight, and tumor volume were conducted as in example 17.
Conclusion: At the dose of 15 μg per mouse, 7G29 and 19G295 can significantly inhibit tumor growth on D15, as shown in
Methods: 3×106 cells/0.1 ml OS-RC-2 cell suspension and 6×106 cells/0.1 ml PBMC cell suspension were fully mixed in a volume ratio of 1:1 and inoculated subcutaneously into NOD/SCID mice aged 5 to 7 weeks weighing 18 to 22 g, 0.2 mL per mouse. The animals were randomly divided into three groups according to the body weight: Model group (PBS, n=6), 18G272 group (30 μg per mouse once daily, n=6), 19G295 group (30 μg per mouse once daily, n=6).
Administration route and administration frequency were the same as in Example 17.
The general clinical observation, body weight, and tumor volume were conducted as in example 17.
Conclusion: Compared with the model group, 18G272 can significantly inhibit the tumor growth of mice on D22 at the dose of 30 μg per mouse. 19G295 had a trend of tumor inhibition on D22, but there was no significant difference compared with the model group. On D30, 18G272 still had a very efficacious inhibition of tumor growth in mice, with a very significant difference. 19G295 had a tendency to inhibit the tumor on D30, but there was no significant difference compared with the model group, as shown in
a Indicates TGI calculated according to tumor volume on day 22 after cell inoculation.
Methods: 3×106 cells/0.1 ml OS-RC-2 cell suspension and 6×106 cells/0.1 ml PBMC suspension were fully mixed in a volume ratio of 1:1 and inoculated subcutaneously into NOD/SCID mice aged 5 to 7 weeks weighing 18 to 22 g, 0.2 mL per mouse. The animals were randomly divided into two groups according to the body weight: Model group (PBS) and 18G272 group (30 μg per mouse once daily). There were six animals in each group.
Administration route and administration frequency were the same as in Example 17.
General clinical observation, body weight, tumor volume, and tumor weight detection were the same as in example 17.
Conclusion: 18G272 can significantly inhibit the tumor growth of mice bearing OS-RC-2 at the dose of 30 μg per mouse, as shown in
a Indicates TGI was calculated according to tumor weight on day 29 after cell inoculation.
Methods: 3×106 cells/0.1 ml OS-RC-2 cell suspension and 6×106 human PBMC suspension were fully mixed in a volume ratio of 1:1 and inoculated subcutaneously into NOD/SCID mice aged 5 to 7 weeks weighing 18 to 22 g, 0.2 mL per mouse. The animals were randomly divided into two groups according to the body weight: Model group (PBS) and 19G294 group (30 μg per mouse once daily). There were 5 animals in each group.
Administration route and administration frequency were the same as in Example 17.
General clinical observation, body weight, and tumor volume were conducted as in example 17.
Conclusion: 19G294 can significantly inhibit the tumor growth of mice bearing OS-RC-2 at the dose of 30 μg per mouse, as shown in
Combined with the results of above four experiments, the efficacy of 19G295 was equivalent to 17G29, while inferior to 18G272 in NOD/SCID mice subcutaneously bearing OS-RC-2 human renal cancer cell xenografts. 19G294 was similar to 18G272, and both could significantly inhibit or even completely inhibit tumor growth of mice bearing OS-RC-2. In terms of safety, 19G295 was similar to 17G29, inferior to 18G272, and 19G294 was similar to 18G272. In conclusion, 18G272 and 19G294 had better efficacy and safety than 17G29 in vivo.
Methods: 3×106 cells/0.1 ml OS-RC-2 cell suspension and 6×106 cells/0.1 ml PBMC suspension were fully mixed in a volume ratio of 1:1 and inoculated subcutaneously into NOD/SCID mice aged 5 to 7 weeks weighing 18 to 22 g, 0.2 mL per mouse. According to the body weight, the animals were randomly divided into five groups: Model group (PBS), 19G294 group (5 μg per mouse once daily), 19G294 group (15 μg per mouse once daily), 19G294 group (30 μg per mouse once daily), 21G587 group (30 μg per mouse once daily). There were three animals in each group.
Administration route and administration frequency were the same as in Example 17.
General clinical observation, body weight, tumor volume, and tumor weight detection were the same as in example 17.
Conclusion: 19G294 (15 μg, 30 μg per mouse) and 21G587 (30 μg per mouse) could significantly inhibit the tumor growth of mice bearing OS-RC-2. The inhibition of tumor growth by 19G294 was in dose-dependent. Mice bearing OS-RC-2 were prone to die or had significant weight loss during the test. Under the conditions of this test, 19G294 (5, 15, 30 μg/mouse) and 21G587 (30 μg/mouse) could improve the decline in health status of animals caused by tumor growth and reduce the mortality of animals, and animals in these groups were well tolerated. The results were shown in
a Indicates TGI calculated according to tumor weight on day 33 after cell inoculation.
Methods: 3×106 cells/0.1 ml OS-RC-2 cell suspension and 6×106 cells/0.1 ml human PBMC suspension were fully mixed in a volume ratio of 1:1 and inoculated subcutaneously into NOD/SCID mice aged 5 to 7 weeks weighing 18 to 22 g, 0.2 mL per mouse. According to the body weight, the animals were randomly divided into 5 groups: Model group (PBS), positive control-A (BAVENCIO, 1.8 mg/mouse once; Axitinib, 30 μg/mouse once), positive control-B (BAVENCIO, 1.8 mg/mouse once; Axitinib, 60 μg/mouse once), 19G294 (30 μg/mouse), YS110 (205 μg/mouse once); There were 3 to 4 animals in each group. Axitinib was administered by gavage. BAVENCIO, test molecules, and PBS were administered by intravenous injection. Axitinib, 19G294 and PBS were administered on the day of inoculation for 5 consecutive days. Two days after drug withdrawal, the second course of treatment was administered (5 consecutive days), once a day (10 doses total). BAVENCIO was administered once a week for 2 consecutive weeks starting on the day of inoculation (2 doses total). YS110 was administered twice a week for 2 weeks starting on the day of inoculation (4 doses total).
General clinical observation, body weight, tumor volume, and tumor weight detection were the same as those of example 17.
Conclusion: 19G294 significantly inhibited tumor growth of mice bearing OS-RC-2 at the dose of 30 μg per mouse. BAVENCIO (1.8 mg/mouse) combined with axitinib (30 μg/mouse, 60 μg/mouse) and YS110 (205 μg/mouse) did not significantly inhibit tumor growth of mice bearing OS-RC-2. Mice bearing OS-RC-2 were prone to die or had significant weight loss during the experimental period. Under the conditions of this experiment, 19G294 (30 μg/mouse) could improve the decline in health status of animals caused by tumor growth and reduce the mortality of animals, and mice in the group were well tolerated during the treatment. Neither BAVENCIO combined with axitinib (two dose groups) nor YS110 had the above improvement effects. In conclusion, 19G294 had better efficacy than BAVENCIO combined with axitinib and YS110 under the conditions of this experiment. The results were shown in
a Indicated that TGI was calculated according to tumor weight on day 31 after cell inoculation.
Methods: 7×106 cells/0.1 ml A498 cell suspension and 7×106 cells/0.1 ml PBMC suspension were fully mixed in a volume ratio of 1:1 and inoculated subcutaneously into NOD/SCID mice aged 5 to 7 weeks weighing 18 to 22 g, 0.2 mL per mouse. According to the body weight, the animals were randomly divided into 5 groups: Model group (PBS), positive control-B (BAVENCIO, 1.8 mg/mouse, Axitinib, 60 μg/mouse), positive control-C (BAVENCIO, 1.8 mg/mouse, Axitinib, 120 μg/mouse), 19G294 (30 μg/mouse), YS110 (205 μg/mouse), There were three animals in each group. Axitinib was administered by gavage. BAVENCIO, test molecules, and PBS were administered by intravenous injection. Axitinib, 19G294 and PBS were administered on the day of inoculation for 5 consecutive days. Two days after drug withdrawal, the second course of treatment was administered (5 consecutive days), once a day (10 doses total). BAVENCIO was administrated once every 2 weeks from the day of inoculation (once in total). YS110 was administered twice a week for 2 weeks starting on the day of inoculation (4 doses total).
General clinical observation, body weight, tumor volume, and tumor weight detection were the same as those of example 17.
Organ weight and organ coefficient: The animals were euthanized after the last examination. The organs of the mice were separated, rinsed with normal saline, blotted dry with filter paper, weighed and photographed. Organ coefficient=organ weight/body weight of mice×100% (organ weight and body weight of mice were measured in g).
Conclusion: 19G294 could significantly inhibit the tumor growth of mice bearing A498 at the dose of 30 μg per mouse. BAVENCIO (1.8 mg/mouse) combined with axitinib (60 μg/mouse, 120 μg/mouse) and YS110 (205 μg/mouse) did not significantly inhibit tumor growth of mice bearing A498. Mice in 19G294 (30 μg/drug), BAVENCIO combined with axitinib (two dose groups) and YS110 group were well tolerated during the treatment. 19G294 (30 μg/mouse) and YS110 had no significant effect on kidney, spleen, liver, lung and corresponding organ coefficient of mice. BAVENCIO combined with axitinib (two dose groups) had a significant effect on liver and/or liver coefficient, but had no significant effect on kidney, spleen, lung and corresponding organ coefficient of mice. In conclusion, under the conditions of this experiment, 19G294 had better efficacy than BAVENCIO combined with axitinib and YS110, and had better safety than BAVENCIO combined with axitinib. The results were shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111245489.5 | Oct 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/126880 | 10/24/2022 | WO |
