ANTI-B7H4 ANTIBODY, AND BISPECIFIC ANTIBODY AND USE THEREOF
20230312722 · 2023-10-05
Assignee
Inventors
- Xiaodong Wu (Shanghai, CN)
- Yongqiang Wang (Shanghai, CN)
- Fei Chen (Shanghai, CN)
- Jinqiu HE (Shanghai, CN)
- Gezi JIA (Shanghai, CN)
- Chuchu ZHAO (Shanghai, CN)
- Qingfang CHEN (Shanghai, CN)
- Yiping Rong (Shanghai, CN)
Cpc classification
G01N33/57484
PHYSICS
G01N33/535
PHYSICS
C07K16/2809
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
C07K2317/732
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
Abstract
Provided are a B7H4-targeting antibody or a variant thereof, and a bispecific antibody containing the B7H4-targeting antibody, wherein the B7H4-targeting antibody comprises a light chain variable region and a heavy chain variable region. The variant has an amino acid mutation in the light chain variable region or heavy chain variable region of the antibody and can maintain the function of the antibody. The B7H4-targeting antibody has the activity of binding to human B7H4 and cynomolgus macaque B7H4, and does not cross-react with other B7 family members. The B7H4-targeting antibody exhibits good anti-tumor activity, T-cell activation activity and internalization activity in in-vivo and in-vitro experiments, and is more suitable for use as an ADC drug. Further provided is a bispecific antibody targeting B7H4 and CD3, which has a longer half-life, retains good stability and hydrophilicity, and reduces toxicity while ensuring efficacy.
Claims
1. A B7H4-targeting antibody or a variant thereof, wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein: the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 47, 54 and 64, respectively; the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 9, 21 and 35, respectively; or the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 48, 54 and 65, respectively; the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 11, 22 and 36, respectively; wherein the variant comprises a light chain variable region and a heavy chain variable region, wherein: the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 47, 54 and 67, respectively; the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 9, 23 and 35, respectively; or the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 47, 54 and 67, respectively; the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 9, 24 and 35, respectively; or the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 47, 54 and 69, respectively; the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 9, 24 and 38, respectively; or the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 48, 55 and 66, respectively; the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 11, 22 and 36, respectively.
2. (canceled)
3. The antibody or the variant thereof according to claim 1, wherein the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 87; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 77; or, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 88; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 79; or, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 89; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 79; or, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 90; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 80; or, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 90; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 81; or, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 93; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 83; or, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 91; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 79.
4. The antibody or the variant thereof according to claim 1, wherein the antibody further comprises a heavy chain constant region and/or a light chain constant region.
5. The antibody or the variant thereof according to claim 1, wherein the antibody is a full-length antibody, an Fab, an Fab′, an F(ab′).sub.2, an Fv, an scFv, or a monoclonal or polyclonal antibody prepared from an antibody as above.
6. The antibody according to claim 5, comprising (1) a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 95; the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 109; or, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 98; the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 112; or, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 98; the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 113; or, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 99; the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 114; or, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 100; the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 114; or, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 101; the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 109; or, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 102; the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 114; or, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 106; the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 117; or, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 98; the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 115; or, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 103; the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 112; or, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 103; the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 115; or, (2) a heavy chain, wherein the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 132.
7. A B7H4-targeting bispecific antibody comprising a protein functional region A and a protein functional region B, wherein the protein functional region A is the B7H4-targeting antibody according to claim 1 and the protein functional region B is an antibody not targeting B7H4.
8-22. (canceled)
23. The bispecific antibody according to claim 7, wherein the antibody not targeting B71H4 is a CD3-targeting antibody.
24. The bispecific antibody according to claim 23, wherein the CD3-targeting antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 46, 53 and 63, respectively; the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 8, 20 and 34, respectively; or the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 46, 53 and 63, respectively; the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 10, 20 and 34, respectively.
25. The bispecific antibody according to claim 24, wherein the CD3-targeting antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 86; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 76; or the light chain variable region VL comprises an amino acid sequence as set forth in SEQ ID NO: 86; the heavy chain variable region VH comprises an amino acid sequence as set forth in SEQ ID NO: 78; or the light chain variable region VL comprises an amino acid sequence as set forth in SEQ ID NO: 86; the heavy chain variable region VH comprises an amino acid sequence as set forth in SEQ ID NO: 84.
26. The bispecific antibody according to claim 7, wherein the protein functional region B comprises a light chain variable region and a heavy chain variable region, and the protein functional region A comprises a light chain variable region and a heavy chain variable region; wherein, in the protein functional region B, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 8, 20 and 34, respectively; in the protein functional region A, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 47, 54 and 64, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 9, 21 and 35, respectively; or, in the protein functional region B, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 8, 20 and 34, respectively; in the protein functional region A, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 47, 54 and 67, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 9, 23 and 35, respectively; or, in the protein functional region B, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 8, 20 and 34, respectively; in the protein functional region A, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 47, 54 and 67, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 9, 24 and 35, respectively; or, in the protein functional region B, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 8, 20 and 34, respectively; in the protein functional region A, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 48, 54 and 65, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 11, 22 and 36, respectively; or, in the protein functional region B, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 10, 20 and 34, respectively; in the protein functional region A, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 47, 54 and 64, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 9, 21 and 35, respectively; or, in the protein functional region B, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 10, 20 and 34, respectively; in the protein functional region A, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 47, 54 and 67, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 9, 23 and 35, respectively; or, in the protein functional region B, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 10, 20 and 34, respectively; in the protein functional region A, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 47, 54 and 67, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 9, 24 and 35, respectively; or, in the protein functional region B, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 10, 20 and 34, respectively; in the protein functional region A, the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 48, 54 and 65, respectively, and the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 with amino acid sequences as set forth in SEQ ID NOs: 11, 22 and 36, respectively.
27. The bispecific antibody according to claim 7, wherein the protein functional region B comprises a light chain variable region and a heavy chain variable region, and the protein functional region A comprises a light chain variable region and a heavy chain variable region; wherein, in the protein functional region B, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 86, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 76; in the protein functional region A, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 87, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 77; or, in the protein functional region B, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 86, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 76; in the protein functional region A, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 90, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 80; or, in the protein functional region B, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 86, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 76; in the protein functional region A, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 90, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 81; or, in the protein functional region B, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 86, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 76; in the protein functional region A, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 91, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 79; or, in the protein functional region B, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 86, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 78; in the protein functional region A, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 87, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 77; or, in the protein functional region B, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 86, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 78; in the protein functional region A, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 90, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 80; or, in the protein functional region B, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 86, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 78; in the protein functional region A, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 90, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 81; or, in the protein functional region B, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 86, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 78; in the protein functional region A, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 91, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 79; or, in the protein functional region B, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 86, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 84; in the protein functional region A, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 91, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 79.
28. The bispecific antibody according to claim 7, wherein the bispecific antibody is selected from the group consisting of: (1) the bispecific antibody comprises three polypeptide chains, wherein: a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 109; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 118; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 119; or, a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 114; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 120; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 119; or, a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 114; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 121; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 119; or, a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 115; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 122; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 119; or, a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 126; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 118; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 109; or, a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 127; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 118; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 109; or, a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 110; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 128; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 129; or, a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 110; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 130; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 129; (2) the bispecific antibody comprises four polypeptide chains, wherein: a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 97; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 123; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 118; a fourth polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 109; or, a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 97; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 123; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 120; a fourth polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 114; or, a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 97; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 123; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 121; a fourth polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 114; or, a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 97; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 123; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 122; a fourth polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 115; or, a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 97; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 124; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 118; a fourth polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 109; or, a first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 97; a second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 125; a third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 118; a fourth polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 109.
29. A chimeric antigen receptor comprising the antibody according to claim 1.
30. A genetically modified cell comprising the chimeric antigen receptor according to claim 29, wherein the cell is an immune cell.
31. An isolated nucleic acid encoding the antibody according to claim 1.
32. An expression vector comprising the isolated nucleic acid according to claim 31.
33. An antibody-drug conjugate comprising an antibody moiety and a conjugate moiety, wherein the antibody moiety comprises the antibody according to claim 1, and the conjugate moiety is a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof, wherein the antibody moiety and the conjugate moiety are conjugated via a chemical bond or a linker.
34. A pharmaceutical composition comprising the antibody according to claim 1 and optionally a pharmaceutically acceptable carrier.
35. A method for the treatment and/or prevention of a cancer in a subject, comprising administrating an effective amount of the antibody according to claim 1 to the subject, wherein the cancer is a B7H4 positive tumor.
36. The method according to claim 35, wherein the cancer is selected from breast cancer, ovarian cancer and endometrioma.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0151] The present invention is further illustrated by the following examples, which are not intended to limit the present invention. Experimental procedures without specified conditions in the following examples are performed in accordance with conventional procedures and conditions, or in accordance with instructions.
Example 1. Acquisition of Anti-B7H4 Antibody Molecules
[0152] Experimental animals, which may be mice, rats, rabbits, sheep and camels, can be immunized with the B7H4 recombinant protein or cells overexpressing B7H4 to obtain antibody molecules specifically binding to B7H4. Typically, the resulting antibody molecules are non-human antibodies. After obtaining non-human antibodies, these molecules need to be humanized by antibody engineering technology to reduce immunogenicity and improve druggability. However, the humanization of antibodies is complex in terms of the technology, and the humanized molecules tend to have reduced affinity for antigens. On the other hand, advances in transgenic technology have made it possible to develop genetically engineered mice that carry a human immunoglobulin immune repertoire and have the endogenous murine immune repertoire deleted. The Harbour H2L2 mice (Harbour Antibodies BV) are transgenic mice that carry human immunoglobulin immune repertoire, and the antibodies generated by the transgenic mice has a fully human sequence, thus eliminating the need for further humanization and greatly improving the efficiency of therapeutic antibody development.
[0153] 1.1 Immunization of Mice
[0154] Harbour H2L2 mice were subjected to multiple rounds of immunization with a soluble recombinant human B7H4-ECD-mFc fusion protein (Sino Biological, #10738-H05H) as an antigen. The antigenic protein was mixed with an immunoadjuvant to form an immunogenic reagent, which was then injected subcutaneously via the groin or intraperitoneally. In each round of immunization, each mouse received a total injection dose of 100 μL. In the first round of immunization, each mouse received the immunization with an immunogenic reagent prepared by mixing 50 μg of antigenic protein with complete Freund's adjuvant (Sigma, #F5881) in a 1:1 volume ratio. In each subsequent round of booster immunization, each mouse received an immunization with an immunogenic reagent prepared by mixing 25 μg of antigenic protein with Sigma Adjuvant System adjuvant (Sigma, #S6322). The interval between rounds of booster immunization was at least two weeks. In general, there are 6-7 rounds of booster immunizations. The immunization was performed at days 0, 14, 28, 42, 56, 70, 84 and 98; and the antibody titer in serum of mice was measured at days 49 and 77. The last round of booster immunization was performed at a dose of 25 μg of antigenic protein per mouse 5 days before the isolation of H2L2 mouse splenic B cells.
[0155] Or plasmids encoding mouse CD40L, after transfected with CHO-K1 cells (CHO-K1/hu B7H4, Harbour BioMed) overexpressing human B7H4, were mixed with an immunoadjuvant to obtain an immunogenic reagent, and the mice were then immunized with 5×10.sup.6 cells per mouse, the immunization process being the same as protein immunization.
[0156] 1.2 Serum Titer Assay
[0157] At specific time points, the sera of mice were collected, and the titer of antibody binding to B7H4 protein in the sera was determined by the ELISA method and the titer of antibody binding to B7H4-overexpressing cells in the sera was determined by the FACS method.
[0158] In the ELISA method, an ELISA plate (coming, 9018) was coated with 1 μg/mL hB7H4-ECD-his protein (Sino Biological, #10738-H08H) at 100 L/well and incubated overnight at 4° C.; after 2 rinses, the plate was blocked with 1% BSA in PBST for 2 hours at 37° C.; the plate was added with serially-diluted sera at 100 μL/well and incubated for 1 hour at 37° C.; after 3 rinses, the plate was added with anti-rat-HRP (sigma, #A5795) diluted at 1:5000 at 100 L/well and incubated for 30 minutes at 37° C. After 3 rinses, the plate was added with TMB substrate at 100 μL/well and incubated for about 10 minutes, and then added with 1N HCl at 50 μL/well for termination of the color development, and then the absorbance at 450 nm was read (Molecular Devices, Plus 384).
[0159] In the FACS method, serially-diluted mouse sera were incubated with HEK293-B7H4 cells for 1 hour at 4° C.; after 2 washes of the cells, a secondary antibody Anti-Rat IgG (H+L) (Life technologies, A11006) was added and incubated for 1 hour at 4° C. and after 2 washes, the cells were resuspended and detected by a flow cytometer (BD, Flibur). HEK293 cells served as background controls.
[0160] 1.3 Screening of Anti-B7H4 Antibodies by Hybridoma Technology
[0161] Immunized mice with high serum titer were selected for one final immunization and then sacrificed. Spleen cells and SP2/0 myeloma cells (ATCC, CRL-1581) were electrofused at a cell ratio of 4:1 with the electrofusion parameters shown as follows: V1: 50V, t1: 15 s, V2: 600 V, t2: 20 μs, t3: 0.5 s, n: 1, t4: 7 s, V+/−: +, and fade: on. The cells were resuspended in a DMEM medium containing 20% FBS and HT at 1×10.sup.5/100 μL/well. After 24 hours, DMEM containing 20% FBS and 2×HT was added at 100 μL/well for further culturing. The supernatant was subsequently collected and detected for the antibody titer. Generally, 9-15 days after fusion, supernatants of protein-immunized mice were taken and subjected to primary screening with Acumen, and detected for the binding to CHO-K1/huB7H4 cells; supernatants of cell-immunized mice were taken and subjected to primary screening with Mirrorball (SPT Labtech, Mirrorball® fluorescence cytometer), and detected for the binding to HEK-293/huB7H4 cells. Positive clones were selected and then confirmed by ELISA and FACS to detect their binding ability to CHO-K1 cell line overexpressing human B7H4 (CHO-K1/huB7H4), CHO-K1 cell line overexpressing cynomolgus monkey B7H4 (CHO-K1/cynoB7H4), and CHO-K1 cell line overexpressing mouse B7H4 (CHO-K1/mB7H4). Positive wells were further subcloned by limiting dilution and further screened by ELISA and FACS methods. Clones with better binding to human and monkey B7H4 are selected for sequencing.
[0162] 1.4. Screening of Anti-B7H4 Antibodies by In Vitro Cloning Technique for B Cells
[0163] The spleens of the mice were removed, ground and filtered through a 200-mesh filter, and the single cell suspensions were sorted according to the mouse memory B cell sorting kit (Miltenyi, #130-095-838). The sorted cells were subjected to immunofluorescence staining.
[0164] B200 positive cells (BioLegend, #103227), IgM negative cells (BioLegend, #406506) and B7H4 specific positive cells (BioLegend, #405207) were sorted using a flow cell sorter S3e. The cells obtained by sorting were cultured in a 96-well cell culture plate at a density of 5 cells per well, and irradiated EL4 cells were previously plated on the cell culture plate as feeder cells.
[0165] After 14 days of culture, culture supernatants were collected and subjected to ELISA assay, and for wells having binding activity to B7H4 protein, cells were taken and subjected to RT-PCR (SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (#634892), I-5™ 2× High-Fidelity Master Mix (#I5HM-5000)). The light and heavy chains obtained by amplification were spliced into an scFv by overlap PCR and expressed in E. coli, the expression supernatants were subjected to ELISA assay, and the positive clones were sequenced.
[0166] 1.5. Sequence Analysis and Sequence Optimization of Anti-B7H4 Antibodies
[0167] The nucleotide sequences encoding the variable domains of the antibody molecules and the corresponding amino acid sequences were obtained through conventional sequencing means. 3 monoclonal sequences were obtained. In this example, the sequences of the variable domains of the anti-B7H4 monoclonal antibody molecules obtained from immunized Harbour H2L2 mice were human antibody sequences, whose germline gene analysis and post-translational modification site (PTM) analysis are listed in Table 1-1.
[0168] Chemical modifications, sometimes introduced after amino acid chains of a protein or polypeptide is translated and synthesized in a cell, are called post-translational modifications (PTMs). For antibodies, some PTM sites are very conservative. For example, the conservative amino acid asparagine (Asn) at position 297 (EU numbering) of the constant domain of the human IgG1 antibody is often glycosylated to form a saccharide chain whose structure is critical for antibody structure and associated effector functions. However, PTMs may have a greater effect on antigen binding or result in changes in the physicochemical properties of the antibody, if they are present in the variable domains, particularly in the antigen binding regions (e.g., CDRs) of an antibody. For example, glycosylation, deamidation, isomerization, oxidation, and the like may increase the instability or heterogeneity of antibody molecules, thereby increasing the difficulty and risk of antibody development. Thus, it is very important for the development of therapeutic antibodies to avoid some potential PTMs. As experience has accumulated, it has been found that some PTMs are highly correlated with the composition of amino acid sequences, especially the “pattern” of the composition of adjacent amino acids, which makes it possible to predict potential PTMs from the primary amino acid sequences of a protein. For example, it can be predicted that there is an N-linked glycosylation site from the N-x-S/T sequence pattern (asparagine at the first position, any amino acid other than non-proline at the second position, and serine or threonine at the third position). The amino acid sequence patterns leading to PTMs may be derived from germline gene sequences, e.g., the human germline gene fragment IGHV3-33 naturally having a glycosylation pattern NST in the FR3 region; or they may also be derived from somatic hypermutations.
[0169] The amino acid sequence patterns of PTMs may be disrupted by amino acid mutations, thereby reducing or eliminating the formation of specific PTMs. There are different methods for designing mutations depending on the antibody sequences and PTM sequence patterns. One method is to replace a “hot spot” amino acid (e.g., N or S in the NS pattern) with an amino acid with similar physicochemical properties (e.g., to mutate N into Q). If the PTM sequence pattern is derived from somatic hypermutations and is not present in the germline gene sequence, the other method can be to replace the sequence pattern with the corresponding germline gene sequence. In practice, a variety of methods for designing mutations may be used for the same PTM sequence pattern.
[0170] The sequences of the new antibody molecules obtained from amino acid mutations on the sequences of antibodies PR001476 and PR002037 are listed in Table 1-2.
TABLE-US-00001 TABLE 1-1 Germline gene analysis and post-translational modification site (PTM) analysis of anti-B7H4 antibodies Recombinant VH germline VL germline Recombinant antibody Clone No. V gene V gene VH PTM VL PTM antibody subtype 80C8-2E9 IGHV3-30 IGKV3-15 DG (HCDR2) NS (LCDR3) PR001476 Human IgG1 1025_B-1H11 IGHV1-69 IGKV3-15 C (LFR3) None PR002037 Human IgG1 1025_B-2A1 IGHV1-69 IGKV3-15 None None PR002038 Human IgG1
TABLE-US-00002 TABLE 1-2 Mutation site designs of sequences of antigen-binding proteins Recombinant Initial Variable region antibody antibody Variant mutations subtype Fc mutation PR001476 PR002405 H: D54E; L: N92Q Human IgG1 PR001476 PR002408 H: G55A; L: N92Q Human IgG1 PR001476 PR002411 Human IgG1 S239D, I332E PR001476 PR002418 H: G55A; L: N92Q Human IgG1 S239D, I332E PR001476 PR003369 H: G55A, G101A; L: N92R Human IgG1 S239D, I332E PR002037 PR002410 L: C87Y Human IgG1 PR002037 PR002420 Human IgG1 S239D, I332E PR002037 PR002421 L: C87Y Human IgG1 S239D, I332E
[0171] 1.6. Affinity Maturation of Antibody PR001476
[0172] The molecule PR001476 was modified by site-directed mutagenesis to improve its affinity for binding to B7H4. This method of affinity maturation is divided into two rounds.
[0173] In the first round, amino acids of the heavy chain CDR3 and light chain CDR3 (as defined by Chothia CDRs) of the molecule PR001476 were scanned point-by-point to create single-site saturation mutagenesis libraries for multiple amino acid positions. Two saturation mutagenesis libraries were screened, positive molecules with a signal of 2-fold over those of wild type were picked out for sequencing and further identified, and a plurality of mutation hot sites were selected according to the binding ability of the positive molecules to human B7H4.
[0174] In the second round, the hot sites found by the saturation mutagenesis in the first round were randomly combined to create a library containing all mutation combinations. The combination library was then screened. Several mutants were selected by sequencing the positive molecules and detecting their binding ability to human B7H4. The selected mutants were represented by the corresponding clone numbers, for example, PR001476-R1-25B3 and PR001476-R1-26D7.
[0175] The mutants were constructed into mammalian expression vectors for the expression and purification of the proteins. The mutants were then detected for their binding ability to B7H4 using FACS and Fortebio Octet. PR003369 in Table 1-2 is a preferred mutant derived from PR001476.
[0176] 1.7. Preparation of Recombinant Antibodies and Physicochemical Property Characterization Analysis
[0177] 1.7.1. Expression and Purification of Antibodies
[0178] This example describes a general method of antibody preparation in mammalian host cells (e.g., human embryonic kidney cell HEK293 or Chinese hamster ovary CHO cells and derived cells thereof) by using such techniques as transient transfection and expression, and affinity capture and separation. This method is applicable to an antibody of interest comprising an Fc region. The antibody of interest may consist of one or more protein polypeptide chains, and may be derived from one or more expression plasmids.
[0179] The amino acid sequences of the polypeptide chains of the antibody were converted into nucleotide sequences by codon optimization. The encoding nucleotide sequences were synthesized and cloned into expression vectors compatible with the host cell. The mammalian host cells were transfected simultaneously with plasmids encoding the polypeptide chains of the antibody in a particular ratio, and the recombinant antibody with correct folding and assembly of polypeptide chains could be obtained by the conventional recombinant protein expression and purification techniques. Specifically, FreeStyle™ 293-F cells (Thermo, #R79007) were expanded in FreeStyle™ F17 Expression Medium (Thermo, #A1383504). Before the transient transfection, the cells were adjusted to a concentration of 6×10.sup.5 cells/mL to 8×10.sup.5 cells/mL, and cultured in a shaker at 37° C. with 8% CO.sub.2 for 24 hours at a concentration of 1.2×10.sup.6 cells/mL. 30 mL of cultured cells were taken. Plasmids encoding the polypeptide chains of the antibody were mixed in a certain ratio, and a total of 30 μg of the plasmids (the ratio of the plasmids to cells was 1 μg:1 mL) were dissolved in 1.5 mL of Opti-MEM reduced serum medium (Thermo, #31985088). The resulting mixture was filtered through a 0.22 μm filter membrane for sterilization. Then, 1.5 mL of Opti-MEM was dissolved in 120 μL of 1 mg/mL PEI (Polysciences, #23966-2), and the mixture was left to stand for 5 minutes. PEI was slowly added to the plasmids, and the mixture was incubated at room temperature for 10 min. The mixed solution of plasmids and PEI was slowly added dropwise while shaking the culture flask, and the cells were cultured in a shaker at 37° C. with 8% CO.sub.2 for 5 days. Cell viability was measured after 5 days. The culture was collected and centrifuged at 3300 g for 10 min, and then the supernatant was collected and centrifuged at high speed to remove impurities. A gravity column (Bio-Rad, #7311550) containing MabSelect™ (GE Healthcare, #71-5020-91) was equilibrated with a PBS buffer (pH 7.4) and rinsed with 2-5 column volumes of PBS. The column was loaded with the supernatant sample, and rinsed with 5-10 column volumes of PBS buffer, followed by 0.1 M glycine at pH 3.5 to elute the target protein. The eluate was adjusted to neutrality with Tris-HCl at pH 8.0, and concentrated and buffer exchanged into PBS buffer or a buffer with other components with an ultrafiltration tube (Millipore, #UFC901024) to obtain a purified solution of the recombinant antibody. Finally, the purified antibody solution was determined for concentration using NanoDrop (Thermo, NanoDrop™ One), subpackaged and stored for later use.
[0180] 1.7.2. Analysis of Protein Purity and Polymers by SEC-HPLC
[0181] In this example, analytical size-exclusion chromatography (SEC) was used to analyze the protein sample for purity and polymer form. An analytical chromatography column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 μm, 7.8 mm×30 cm) was connected to a high-pressure liquid chromatograph HPLC (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with a PBS buffer at room temperature for at least 1 h. A proper amount of the protein sample (at least 10 μg) was filtered through a 0.22 μm filter membrane and then injected into the system, and an HPLC program was set: the sample was passed through the chromatography column with a PBS buffer at a flow rate of 1.0 mL/min for a maximum of 25 minutes. An analysis report was generated by the HPLC, with the retention time of the components with different molecular sizes in the sample reported.
[0182] 1.7.3. Protein Purity and Hydrophobicity Analysis by HIC-HPLC
[0183] Analytical hydrophobic interaction chromatography (HIC) was used to analyze the protein sample for purity and hydrophobicity. An analytical chromatography column TSKgel Butyl-NPR (Tosoh Bioscience, 14947, 4.6 mm×3.5 cm) was connected to a high-pressure liquid chromatograph HPLC (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with a PBS buffer at room temperature for at least 1 hour. The method consisted of a linear gradient from 100% mobile phase A (20 mM histidine, 1.8 M ammonium sulfate, pH 6.0) to 100% mobile phase B (20 mM histidine, pH 6.0) within 16 minutes, wherein the flow rate was set at 0.7 mL/min, the sample concentration was 1 mg/mL, the injection volume was 20 μL, and the detection wavelength was 280 nm. After being recorded, the chromatogram was integrated using ChemStation software and relevant data were calculated. An analysis was generated, with the retention time of the components with different molecular sizes in the sample reported.
[0184] 1.7.4. Determination of Thermostability of Protein Molecules by DSF
[0185] Differential scanning fluorimetry (DSF) is a commonly used high-throughput method for determining the thermostability of proteins. In this method, changes in the fluorescence intensity of the dye that binds to unfolded protein molecules were monitored using a real-time quantitative fluorescence PCR instrument to reflect the denaturation process of the protein and thus to reflect the thermostability of the protein. In this example, the thermal denaturation temperature (Tm) of a protein molecule was measured by DSF. 10 μg of protein was added to a 96-well PCR plate (Thermo, #AB-0700/W), followed by the addition of 2 μL of 100× diluted dye SYPRO™ (Invitrogen, #2008138), and then the mixture in each well was brought to a final volume of 40 μL by adding buffer. The PCR plate was sealed, placed in a real-time quantitative fluorescence PCR instrument (Bio-Rad CFX96 PCR System), and incubated at 25° C. for 5 min, then at a temperature gradually increased from 25° C. to 95° C. at a gradient of 0.2° C./0.2 min, and at a temperature decreased to 25° C. at the end of the test. The FRET scanning mode was used and data analysis was performed using Bio-Rad CFX Maestro software to calculate the Tm of the sample.
[0186] 1.8. Preparation of Anti-B7H4 Fully Human Recombinant Antibodies
[0187] Anti-B7H4 fully human IgG antibodies obtained in 1.3-1.6 and the optimized antibodies were prepared and analyzed using the method as described in 1.7.1. The results of transient expression and purification for small and large volumes are listed in Table 1-3 and Table 1-4, respectively. In addition, the sequences of anti-B7H4 antibodies (Table 1-5) were obtained from the prior documents and taken as controls in subsequent experiments.
TABLE-US-00003 TABLE 1-3 Expression of anti-B7H4 antibodies Expression system Yield after first SEC-HPLC Antibody and volume purification (mg/L) purity (%) PR001476 HEK293-F (50 mL) 80.6 99.18 PR002037 HEK293-F (30 mL) 17.3 100 PR002038 HEK293-F (30 mL) 34.0 100 PR002408 HEK293-6E (40 mL) 37.0 99.03 PR002411 HEK293-6E (40 mL) 64.8 99.3 PR002418 HEK293-F (1.5 mL) 26.7 n.d. PR003369 HEK293-F (30 mL) 6.6 100 PR002410 HEK293-6E (40 mL) 173.5 98.65 PR002420 HEK293-6E (40 mL) 5.7 99.16 PR002421 HEK293-F (1.5 mL) 73.3 n.d.
TABLE-US-00004 TABLE 1-4 Expression of anti-B7H4 antibodies Expression system Yield after two-step SEC-HPLC Antibody and volume purification (mg/L) purity (%) PR002418 Expi-CHOs (1000 mL) 229 99.45% PR002421 Expi-CHOs (1000 mL) 276.9 99.01% PR003369 Expi-CHOs (1000 mL) 111.7 99.09% Control Expi-CHOs (1000 mL) 180.8 95.17% antibody 2
TABLE-US-00005 TABLE 1-5 Information relating to control antibodies Control Antibody antibodies No. Description Control PR000157 Anti-B7H4 IgG1 antibody (from Patent antibody 1 WO2016040724A1), also known as 1D11.v1.9varC2 Control PR002962 Anti-B7H4 IgG1 antibody (from Patent antibody 2 WO2019040780A1), with site mutation on the Fc region on the basis of PR002961 to enhance ADCC Control PR002961 Anti-B7H4 IgG1 antibody (from Patent antibody 3 WO2019040780A1)
[0188] 1.9. Anti-B7H4 Antibody Sequences and Numbers
[0189] In the present invention, the amino acid sequences of the listed CDRs are shown according to the Chothia scheme. However, it is well known to those skilled in the art that the CDRs of an antibody can be defined in the art using a variety of methods, such as the Kabat scheme based on sequence variability (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health (U.S.), Bethesda, Maryland (1991)), and the Chothia scheme based on the location of the structural loop regions (see J Mol Biol 273: 927-48, 1997). In the technical solution of the present invention, the Combined scheme comprising the Kabat scheme and the Chothia scheme can also be used to determine the amino acid residues in a variable domain sequence. The Combined scheme combines the Kabat scheme with the Chothia scheme to obtain a larger range. See Table 1-6 for details. It will be understood by those skilled in the art that unless otherwise specified, the terms “CDR” and “complementary determining region” of a given antibody or a region (e.g., variable region) thereof are construed as encompassing complementary determining regions as defined by any one of the above known schemes described herein. Although the scope claimed in the present invention is the sequences shown based on the Chothia scheme, the amino acid sequences corresponding to the other schemes for numbering CDRs shall also fall within the scope of the present invention.
TABLE-US-00006 TABLE 1-6 The scheme for numbering the CDRs of the antibody of the present application Kabat Chothia Combined LCDR1 L24--L34 L24--L34 L24-L34 LCDR2 L50--L56 L50--L56 L50-L56 LCDR3 L89--L97 L89--L97 L89-L97 HCDR1 H31--H35 H26--H32 H26-H35 HCDR2 H50--H65 H52--H56 H50-H65 HCDR3 H95--H102 H95--H102 H95-H102
[0190] Laa-Lbb can refer to an amino acid sequence from position aa (the Chothia scheme) to position bb (the Chothia scheme) beginning at the N-terminus of the light chain of the antibody; and Haa-Hbb can refer to an amino acid sequence from position aa (the Chothia scheme) to position bb (the Chothia scheme) beginning at the N-terminus of the heavy chain of the antibody. For example, L24-L34 can refer to the amino acid sequence from position 24 to position 34 according to the Chothia scheme beginning at the N-terminus of the light chain of the antibody; H26-H35 can refer to the amino acid sequence from position 26 to position 35 according to the Chothia scheme beginning at the N-terminus of the heavy chain of the antibody. It should be known to those skilled in the art that there are positions where insertion sites are present in numbering CDRs with the Chothia scheme (see http://bioinf.org.uk/abs/).
[0191] The sequence numbers of the CDRs, variable regions and light and heavy chains corresponding to the sequences of the anti-B7H4 antibodies of the present invention and the control antibody molecules are listed in Table 1-7. PR003366 is a single-chain variable region (scFv) homodimer molecule (scFv-Fc structure) constructed using the variable region sequence of PR002410.
TABLE-US-00007 TABLE 1-7 Sequence numbers of anti-B7H4 antibodies Antibody Light Heavy No. chain chain VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 PR000157 108 94 85 75 45 52 62 7 19 33 PR002961 116 104 92 82 48 54 68 12 25 37 PR002962 116 105 92 82 48 54 68 12 25 37 PR001476 109 95 87 77 47 54 64 9 21 35 PR002037 112 98 88 79 48 54 65 11 22 36 PR002038 113 98 89 79 48 55 66 11 22 36 PR002405 114 99 90 80 47 54 67 9 23 35 PR002408 114 100 90 81 47 54 67 9 24 35 PR002411 109 101 87 77 47 54 64 9 21 35 PR002418 114 102 90 81 47 54 67 9 24 35 PR003369 117 106 93 83 47 54 69 9 24 38 PR002410 115 98 91 79 48 54 65 11 22 36 PR002420 112 103 88 79 48 54 65 11 22 36 PR002421 115 103 91 79 48 54 65 11 22 36 PR003366 132 91 79 48 54 65 11 22 36
[0192] The sequence numbers of the framework regions and Fv corresponding to the sequences of the anti-B7H4 antibodies of the present invention and the control antibody molecules are listed in Table 1-8.
TABLE-US-00008 TABLE 1-8 Sequence numbers of framework regions and Fv of anti-B7H4 antibodies Antibody No. Chain type Fv FWR1 FWR2 FWR3 FWR4 PR000157 Heavy chain 75 1 13 26 39 Light chain 85 40 49 56 70 PR002961 Heavy chain 82 6 18 31 39 Light chain 92 42 51 58 72 PR002962 Heavy chain 82 6 18 31 39 Light chain 92 42 51 58 72 PR001476 Heavy chain 77 3 15 28 39 Light chain 87 42 51 58 72 PR002037 Heavy chain 79 5 17 30 39 Light chain 88 43 51 59 73 PR002038 Heavy chain 79 5 17 30 39 Light chain 89 44 51 60 74 PR002405 Heavy chain 80 3 15 28 39 Light chain 90 42 51 58 72 PR002408 Heavy chain 81 3 15 28 39 Light chain 90 42 51 58 72 PR002411 Heavy chain 77 3 15 28 39 Light chain 87 42 51 58 72 PR002418 Heavy chain 81 3 15 28 39 Light chain 90 42 51 58 72 PR003369 Heavy chain 83 3 15 28 39 Light chain 93 42 51 58 72 PR002410 Heavy chain 79 5 17 30 39 Light chain 91 43 51 61 73 PR002420 Heavy chain 79 5 17 30 39 Light chain 88 43 51 59 73 PR002421 Heavy chain 79 5 17 30 39 Light chain 91 43 51 61 73 PR003366 Heavy chain 79 5 17 30 39 Light chain 91 43 51 61 73 PR000627 Heavy chain 76 2 14 27 39 Light chain 86 41 50 57 71 PR001924 Heavy chain 78 4 16 29 39 Light chain 86 41 50 57 71 PR001848 Heavy chain 78 4 16 29 39 Light chain 86 41 50 57 71 PR003886 Heavy chain 84 2 14 32 39 Light chain 86 41 50 57 71
[0193] The CDR sequences corresponding to the sequences of the anti-B7H-4 antibodies of the present invention and the control antibody molecules are listed in Table 1-9.
TABLE-US-00009 TABLE 1-9 CDR sequences of anti-B7H4 antibodies No. HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 PR000157 GYTFTSY YPGGGY LAGSSYRGAMDS KASQGENKYVA YTSTLQP LQYGDLLYA PR002961 GGSIKSGSY YYSGS EGSYPNQFDP RASQSVSSNLA GASTRAT QQYHSFPFT PR002962 GGSIKSGSY YYSGS EGSYPNQFDP RASQSVSSNLA GASTRAT QQYHSFPFT PR001476 GFTFRSF SYDGSN GGGLRWYFAY RASQSISSNLG GASTRAT QQYNSWPPLT PR002037 EDTFSSY APIFGT GGPYFDY RASQSVSSNLA GASTRAT QQYKNWPFT PR002038 EDTFSSY APIFGT GGPYFDY RASQSVSSNLA GDYIRAT QQYVDLPIT PR002405 GFTFRSF SYEGSN GGGLRWYFAY RASQSISSNLG GASTRAT QQYQSWPPLT PR002408 GFTFRSF SYDASN GGGLRWYFAY RASQSISSNLG GASTRAT QQYQSWPPLT PR002411 GFTFRSF SYDGSN GGGLRWYFAY RASQSISSNLG GASTRAT QQYNSWPPLT PR002418 GFTFRSF SYDASN GGGLRWYFAY RASQSISSNLG GASTRAT QQYQSWPPLT PR003369 GFTFRSF SYDASN GGALRWYFAY RASQSISSNLG GASTRAT QQYRSWPPLT PR002410 EDTFSSY APIFGT GGPYFDY RASQSVSSNLA GASTRAT QQYKNWPFT PR002420 EDTFSSY APIFGT GGPYFDY RASQSVSSNLA GASTRAT QQYKNWPFT PR002421 EDTFSSY APIFGT GGPYFDY RASQSVSSNLA GASTRAT QQYKNWPFT PR003366 EDTFSSY APIFGT GGPYFDY RASQSVSSNLA GASTRAT QQYKNWPFT PR000627 GFTFNTY RSKYNNYA HGNFGNSYVSWFAY RSSTGAVTTSNYAN GTNKRAP ALWYSNLWV PR001924 GFTFSTY RSKYNNYA HGNFGNSYVSWFAY RSSTGAVTTSNYAN GTNKRAP ALWYSNLWV PR001848 GFTFSTY RSKYNNYA HGNFGNSYVSWFAY RSSTGAVTTSNYAN GTNKRAP ALWYSNLWV PR003886 GFTESTY RSKYNNYA HGNFGNSYVSWFAY RSSTGAVTTSNYAN GTNKRAP ALWYSNLWV
Example 2. Detection of Binding Ability of Anti-B7H4 Antibodies to B7H4 by FACS
[0194] This example is intended to investigate the in vitro binding activity of anti-human B7H4 H2L2 monoclonal antibody to human/cynomolgus monkey/mouse B7H4. Antibody binding experiments at the cellular level were performed using CHOK1 cell line overexpressing human B7H4 (CHOK1/hu B7H4, Harbour BioMed), CHOK1 cell line overexpressing cynomolgus monkey B7H4 (CHOK1/cyno B7H4, Harbour BioMed), CHOK1 cell line overexpressing mouse B7H4 (CHOK1/m B7H4, Harbour BioMed) and SK-BR-3 cell line (ATCC® HTB-30) highly expressing human B7H4. Briefly, CHOK1/hu B7H4 cells, CHOK1/cyno B7H4 cells, CHOK1/m B7H4 cells or SK-BR-3 cells were digested and resuspended with PBS containing 2% BSA. The cell density was adjusted to 1×10.sup.6 cells/mL. The cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 μL/well, followed by the addition of test antibodies diluted in a 3-fold concentration gradient of 2 times the final concentration, each at 100 μL/well. The cells were incubated at 4° C. for 2 h away from light. Thereafter, the cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% BSA, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Then 100 μL of a fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-098, 1:500 diluted) was added to each well. The plate was incubated away from light at 4° C. for 1 h. The cells in each well were washed twice with 100 μL of pre-cooled PBS containing 2% BSA, and centrifuged at 500 g at 4° C. for 5 minutes, and then the supernatant was discarded. Finally, the cells in each well were resuspended with 200 μL of pre-cooled PBS containing 2% BSA, and the fluorescence signal values were read using an ACEA Novocyte3000 flow cytometer.
[0195] The antibodies binding to human B7H4, cynomolgus monkey B7H4 and mouse B7H4 on the cell surface and to B7H4 on the surface of tumor cells SK-BR-3 are summarized below (Table 2-1, Table 2-2 and Table 2-3). The variant PR003369 with affinity maturation showed a significant improvement in its binding to tumor cells compared with PR002418 (Table 2-3).
TABLE-US-00010 TABLE 2-1 Anti-B7H4 antibodies (initial antibodies) binding to human B7H4, cynomolgus monkey B7H4 and mouse B7H4 on cell surface and to B7H4 on surface of tumor cells SK-BR-3 CHOK1/hu CHOK1/cyno CHOK1/m B7H4 B7H4 B7H4 SK-BR-3 Maximum EC.sub.50 Maximum EC.sub.50 Maximum EC.sub.50 Maximum EC.sub.50 Antibody MFI (nM) MFI (nM) MFI (nM) MFI (nM) PR002037 1677000 3.196 1207000 3.301 750723 2.698 290692 3.527 PR002038 1146000 20.04 677326 24.76 ~ ~ 82832 85.62 PR001476 1412000 0.7552 971785 0.8861 230031 3.018 277576 0.4706 Control 1839000 1.943 1317000 2.057 857700 1.381 316232 1.175 antibody 1
TABLE-US-00011 TABLE 2-2 Anti-B7H4 antibodies (variant molecules) binding to cynomolgus monkey B7H4 and mouse B7H4 on cell surface and to B7H4 on surface of tumor cells SK-BR-3 CHOK1/cyno B7H4 CHOK1/m B7H4 SK-BR-3 Maximum EC.sub.50 Maximum EC.sub.50 Maximum EC.sub.50 Antibody MFI (nM) MFI (nM) MFI (nM) RP001476 1822000 1.097 353378 3.614 238962 0.367 PR002408 1852000 1.029 398460 1.678 248060 0.3531 PR002418 1790000 0.7985 398407 1.306 251781 0.3112 PR002037 1922000 2.382 1686000 2.57 256727 2.22 PR002410 1862000 2.077 1554000 2.385 245482 1.872 PR002421 1873000 2.048 1465000 1.988 242474 1.92 Control 1986000 2.095 1749000 1.728 265713 1.199 antibody 1
TABLE-US-00012 TABLE 2-3 Binding of PR003369 and PR002418 to tumor cells SK-BR-3 Antibody EC.sub.50 (nM) Maximum MFI PR002418 1.116 162162 PR003369 0.4342 170835
[0196] The results of the initial antibodies binding to human B7H4 on the cell surface are shown in
Example 3. ADCC Activity Assay
[0197] This example is intended to investigate the activity of anti-human B7H4 H2L2 monoclonal antibody in mediating NK cell killing of target cells through the ADCC effect in vitro. In this experiment, the human PBMC was used as an effector cell and cell lines SK-BR-3 and MDA-MB-468 highly expressing B7H4 and a cell line HCC-1954 moderately expressing B7H4 were used as target cells. The killing efficiency was reflected by the conductivity of the target cell measured using an RTCA instrument from ACEA. A 96-well plate E-plate was first equilibrated with 50 μL of complete medium. SK-BR-3, MDA-MB-468 or HCC-1954 cells were digested, resuspended in RPM1640 complete medium containing 10% fetal bovine serum and diluted to 4×10.sup.5/mL. The cell suspension was plated on the 96-well E-plate at 50 μL/well, i.e., 2×10.sup.4/well, and incubated overnight at 37° C. The next day, 50 μL of fresh culture medium containing 2×10.sup.5 PBMCs was added to each well, followed by the addition of 50 μL of antibodies diluted in a 4× concentration gradient with a maximum final concentration of 10 nM. A total of 8 concentrations were set in duplicate for each antibody. The conductivity of the target cells was measured in real time. Generally, the value at hour 4 was used to calculate the target cell killing efficiency=(1−sample/blank control)×100%.
[0198] The ADCC killing activity of PR001476 and its PTM variant/DE mutant antibody against tumor cells SK-BR-3 is shown in
Example 4. Detection of Activation Activity of T Cells
[0199] To detect the function of the anti-B7H4 antibodies for blocking immune checkpoints of T cells and thus activating T cells, in this experiment, full-length B7H4 and anti-human CD3 antibody OKT3 in the form of scFv were overexpressed on HEK293T cells and taken as artificial antigen presenting cells (HEK 293T/OS8/hB7H4, KYinno), a human T cell isolation kit (Miltenyi, #130-096-535) was used to isolate T cells according to the method of the instruction, the artificial antigen presenting cells and the T cells were co-cultured, and the activation effect of the anti-B7H4 antibodies on T cells was detected. Specifically, HEK293T-OS8-hB7H4 was plated at a density of 1×10.sup.4/well and incubated overnight. Human primary T cells were isolated and added to HEK293T/OS8/hB7H4 cells at a density of 2×10.sup.5/100 μL/well, followed by the addition of test antibodies diluted in a 5-fold concentration gradient of 2 folds the final concentration, each at 100 μL/well, wherein the maximum final concentration of the antibody was 10 nM. A total of 6 concentrations were set in duplicate for each antibody. After 3 days of culture, the supernatant was collected and the concentration of IFN-γ was detected by ELISA method. The results showed that PR003369, PR002418, PR002421 and control antibodies all could promote the activation of T cells, wherein the variant PR003369 with affinity maturation had stronger T cell activation activity compared with the PTM variant PR002418 and the control antibody 2, and the mechanism of action of PR003369 may be blocking the interaction between B7H4 with its unknown receptor on T cells. The results of the anti-B7H4 antibodies blocking the immunosuppressive signal of B7H4 to activate T cells are shown in
Example 5. Antibody Internalization Experiment
[0200] Internalization of antibodies was detected using Zenon pHrodo iFL IgG Labeling Reagents kit (Invitrogen, #Z25611). The reagent is a secondary antibody with a fluorescent dye, does not emit fluorescence at neutral pH, can automatically emit bright fluorescence in an acidic pH environment after being combined with the primary antibody and internalized into lysosomes along with the antibody, and can be detected by FACS method. The specific method was as follows: SK-BR-3 cells were collected and centrifuged to discard the supernatant, and the cells were resuspended in a medium to adjust the cell concentration to 3×10.sup.6/mL. The cell suspension was added to a 96-well plate at 50 μL/well, and then incubated overnight in a 37° C. thermostatic incubator. Test antibodies of 4× were prepared at a maximum concentration of 40 nM (4×), and diluted 3-fold for a total of 8 dilutions. A Zenon solution of 4× was prepared, and 25 μL of the test antibody and 25 μL of a Zenon labeling solution of 4× were mixed together and left to stand at room temperature for 5 minutes. Then 50 μL of the labeled antibody was added to a 96-well plate containing the cells, and incubated in a 37° C. thermostatic incubator for 24 hours. The cells were digested and fluorescence values were read on a flow cytometer. The results showed that PR003369 had the highest internalization activity compared with the control antibody RP000014 and other antibodies. The results of internalization of the anti-B7H4 antibodies on SK-BR-3 cells are shown in
[0201] Internalization of the antibodies was detected using a-HFc-CL-MMAF reagent (Moradec, #AH-102-AF). The reagent is a secondary antibody carrying a toxic group MMAF, and the mechanism of the reagent being combined with the primary antibody and internalized along with the antibody to release the toxic group in the cells to kill the target cells is similar to that of ADC. The specific method was as follows: SK-BR-3 cells were collected and centrifuged to discard the supernatant, and the cells were resuspended in medium to a cell concentration of 1×10.sup.5/mL. The cell suspension was added to a 96-well plate at 50 μL/well, and then incubated overnight in a 37° C. thermostatic incubator. Test antibodies of 4× were prepared at a maximum concentration of 40 nM (4×) in 5-fold dilutions for a total of 8 dilutions. An MMAF solution of 4× (4 μg/mL) was prepared. 25 μL of the test antibody of 4× and 25 μL of MMAF solution of 4× were added to a 96-well plate containing cells, and incubated at a 37° C. thermostatic incubator for 72 hours. 100 μL of CTG solution was added to the wells, and the luminescence signals of CTG were read using a microplate reader. The results in
Example 6. Antibody-Drug Conjugate (ADC)
[0202] In this example, an ADC was prepared using ADC conjugation technology by crosslinking a toxic group MMAE to an anti-B7H4 antibody (PR003369 antibody or control antibody 1). The purity parameters of the product are as follows, and the HPLC detection method is the same as that in 1.7.2.
TABLE-US-00013 TABLE 6-1 ADC preparation of anti-B7H4 H2L2 antibody Concen- Antibody tration Storage SEC-HPLC Endotoxin ADC (mg/mL) DAR solution Purity % EU/mg PR003369- 2.66 4.4 PBS, pH 7.2 97.05 <0.06 MMAE Control 2.65 4.1 PBS, pH 7.2 97.42 0.1 antibody 1-MMAE
[0203] To investigate whether the binding activity of the antibody to the B7H4 target was affected after crosslinking of MMAE groups, antibody binding experiments at the cellular level were performed using the cell line MDA-MB-468 highly expressing human B7H4, and the method was the same as that in Example 2. The results in
[0204] To investigate whether the internalization activity of the antibody was affected after crosslinking of MMAE groups, the internalization of the antibody was detected using Zenon pHrodo iFL IgG Labeling Reagents kit (Invitrogen, #Z25611). The experimental method was the same as that in Example 5. The results in
[0205] This example is to investigate the cell killing activity of the antibody with MMAE groups crosslinking ADCs. The cell line MDA-MB-468 highly expressing B7H4 was taken as a target cell, and the killing efficiency was reflected by the conductivity of the target cell measured using an RTCA instrument from ACEA. A 96-well plate E-plate was first equilibrated with 50 μL of complete medium. MDA-MB-468 cells were digested, resuspended in RPM1640 complete medium containing 10% fetal bovine serum and diluted to 1×10.sup.5/mL. The cell suspension was plated on the 96-well E-plate at 50 μL/well, i.e., 5×10.sup.3/well, and incubated overnight at 37° C. The next day, 100 μL of antibodies diluted in a 2× concentration gradient was added to each well with a maximum final concentration of 50 nM. A total of 8 concentrations were set in duplicate for each antibody. The conductivity of the target cells was measured in real time. Generally, the value at hour 96 was used to calculate the target cell killing efficiency=(1−sample/blank control)×100%. The results in
Example 7. Determination of Affinity of Anti-B7H4 Antibodies to Human B7H4 Recombinant Protein
[0206] 7.1. Determination of Affinity by SPR Method
[0207] 10×HBS-EP+ (GE Healthcare, #BR-1006-69) was diluted 10-fold and then taken as an experimental buffer. The flow rate was set at 10 μL/min, and Protein A was coupled to 4 channels of a chip CM5 (GE Healthcare, #BR-1005-30) through the following procedures: 1) the injection time was set to 800 s, and a fresh mixture of 50 mM NHS and 200 mM EDC was injected into the 4 channels in a volume ratio of 1:1; 2) Protein A was diluted to 20 μg/mL with sodium acetate (GE Healthcare, #BR-1003-50) at pH 4.5 and injected into each channel for 800 s; and 3) 1 M ethanolamine at pH 8.5 was injected for 800 s to block the remaining active carboxyl groups on the chip surface. After blocking, the instrument was equilibrated with 1×HBS-EP+buffer for 2 hours, and the final coupling level of Protein A was about 2000 RU.
[0208] A multi-cycle kinetics mode was set at Biacore T200, and each cycle included antibody capture, analyte binding and chip regeneration. Antibodies PR002418 and PR002421, control antibody 1, and control antibody 2 were all diluted to 1 μg/mL, injected into channels 2, 3 and 4 at a flow rate of 10 μL/min for 30 s, and each antibody was captured by a pre-coupled Protein A at level 160 RU. Human B7-H4 (Sino biological, #10738-H08H) was injected into four channels (for control antibody 1, one maximum concentration of 100 nM was added) with a concentration gradient of 0 nM, 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM and 50 nM sequentially, and the flow rate was set at 30 μL/min. The dissociation time was set to 200 s for PR002418, PR002421 and control antibody 2, and 500 s for control antibody 1, with 180 s for each injection. Finally, 10 mM glycine-hydrochloric acid at pH 1.5 (GE Healthcare, #BR-1003-54) was injected at the same flow rate for 30 s to regenerate the chip.
[0209] The experimental results were analyzed using the Biacore T200 analysis software 2.0, with channel 1 subtracted as a reference channel and a 1:1 kinetic fitting model selected as an analysis model. The results are shown in Table 7-1 and A-D of
TABLE-US-00014 TABLE 7-1 Affinity of anti-B7H4 antibodies for binding to human B7H4 protein (SPR method) Antibody concentration ka kd KD Antibody Antigen (nM) (1/Ms) (1/s) (M) PR002418 Human B7H4 (his tag) 50-1.5625 2.01E+05 4.75E−03 2.37E−08 PR002421 Human B7H4 (his tag) 50-1.5625 3.50E+05 3.22E−04 9.21E−10 Control Human B7H4 (his tag) 50-1.5625 2.61E+05 1.16E−03 4.45E−09 antibody 2 Control Human B7H4 (his tag) 100-3.125 4.04E+04 1.89E−04 4.67E−09 antibody 1
[0210] 7.2. Determination of Affinity by BLI Method
[0211] 10× kinetics buffer (ForteBio, #18-1105) was diluted to 1× kinetics buffer for affinity assay and dilution of antigens and antibodies. The binding kinetics between the antigen and the antibody was analyzed by the Biolayer Interferometry (BLI) technique using an Octet Red 96e molecular interaction analyzer (Fortebio).
[0212] When the affinity of the antigen for the antibody was determined, the rotation speed of the sensor was set at 1000 rpm/min. The AHC sensors (Fortebio, #18-5060) placed in a row were equilibrated for 10 minutes in a test buffer before the AHC sensors were used to capture the B7-H4 antibodies at a capture height of 0.7 nm; the AHC sensors, after equilibrated in the buffer for 120 s, bound to 2-fold serially diluted human B7-H4 (concentrations were 50 nM-3.125 nM and 0 nM) for 180 s, followed by dissociation for 300 s. Finally, the AHC sensor was immersed in a 10 mM glycine-hydrochloric acid solution at pH 1.5 for regeneration to elute the proteins bound to the sensor.
[0213] When data analysis was performed using Octet Data Analysis software (Fortebio, version 11.0), 0 nM was taken as a reference well, and reference subtraction was performed; the “1:1 Global fitting” method was selected to fit the data, and the kinetics parameters of the binding of antigens to antigen-binding proteins were calculated, with k.sub.on (1/Ms) values, k.sub.dis (1/s) values and K.sub.D(M) values obtained (see Table 7-2). The results showed that the variant PR003369 with affinity maturation had stronger protein affinity than PR002418.
TABLE-US-00015 TABLE 7-2 Affinity of anti-B7H4 antibodies for binding to human B7H4 protein (BLI method) Concentration KD kon kdis Antibody (nM) (M) (1/Ms) (1/s) Full R{circumflex over ( )}2 PR002418 50-12.5 1.03E−08 2.75E+05 2.84E−03 0.9926 PR003369 50-12.5 1.39E−09 2.83E+05 3.94E−04 0.9981
Example 8. Determination of Epitope Competition of Anti-B7H4 Antibodies Binding to B7H4 by BLI Method
[0214] Epitope competition experiments were performed on B7-H4 antibodies PR002418 and PR002421, control antibody 1 and control antibody 2 by using ForteBio Octet Red96e platform, and the experimental buffer was the same as described above. Step one, acquisition of 100% signal of antibodies: B7-H4 (Acro Biosystems, #B74-H82E2-200 μg) was captured at a capture height of 0.25 nm using an SA sensor (Fortebio, #18-5019). The sensor was equilibrated in a buffer for 120 s and then immersed in each antibody diluted to 100 nM for 240 s, and the final signal of the antibody binding to B7-H4 was recorded as the 100% signal of the antibody. Step two, epitope competition experiment: B7-H4 was captured using an SA sensor at a capture height of 0.25 nm. The sensor was immersed in a primary antibody (at a concentration of 100 nM) for 240 s, and then the SA sensor was immersed in a mixture of the primary and secondary antibodies (both at a final concentration of 100 nM) for 240 s, and the difference in signals after immersion of the sensor in the antibody mixture was recorded as the signal of the antibody as the secondary antibody. The inhibition rate was calculated according to the following formula:
Inhibition (%)=(A−B)/A×100
[0215] A: 100% signal of an antibody (obtained from step one), B: the signal of the antibody as the secondary antibody (obtained from step two).
[0216] If the inhibition rate obtained was greater than 85(%), it means that the epitopes of the two antibodies were completely overlapped; if the inhibition rate was less than 85(%), it means that the epitopes to which the two antibodies bind were not completely overlapped.
[0217] The results in Table 8-1 showed that the epitopes of PR002418 and PR002421 binding to B7-H4 were different, and also different from the epitopes of control antibody 1 and control antibody 2, wherein PR002418 bound to one unique epitope (the first epitope), PR002421 bound to another epitope (the second epitope), and control antibody 1 and control antibody 2 bound to the same epitope (the third epitope).
TABLE-US-00016 TABLE 8-1 Competitive analysis for antibodies binding to B7H4 2nd Ab Competitive inhibition Control Control rate (%) PR002418 PR002421 antibody 2 antibody 1 1st Ab PR002418 94.91 7.23 8.15 11.19 PR002421 7.12 94.77 10.62 10.78 Control 4.49 6.32 95.75 96.57 antibody 2 Control 4.55 1.97 94.58 95.31 antibody 1
Example 9. Cross-Reactivity with Other Members of B7 Family
[0218] The proteins of the B7 family (see Table 9-1 for details) were each diluted to 1 μg/mL with PBS, added to a 96-well plate (Corning, #9018) at 100 μL per well, and incubated overnight at 4° C. After the liquid was discarded, the plate was washed 3 times with PBST buffer (pH 7.4, containing 0.05% tween-20), and 250 μL of 2% BSA blocking buffer was added. The plate was incubated at 37° C. for 1 hour. The blocking buffer was discarded, and the plate was washed 3 times with PBST buffer (pH 7.4, containing 0.05% Tween-20). The test antigen-binding protein was diluted to 2 concentrations: 10 nM and 1 nM, and added at 100 μL per well. The plate was incubated at 37° C. for 1 hour. An isotype antibody was taken as a control. After the plate was washed 3 times with PBST buffer (pH 7.4, containing 0.05% Tween-20), the plate was added with a 5000-fold diluted goat anti-human F(ab′).sub.2 HRP secondary antibody (Jackson ImmunoResearch, 109-035-097), and incubated at 37° C. away from light for 1 hour. After the plate was washed 3 times with PBST buffer (pH 7.4, containing 0.05% Tween-20), TMB (Biopanda, #TMB-S-003) was added at 100 μL/well. The plate was left away from light at room temperature for about 30 minutes. The reactions were terminated by adding 50 μL of stop buffer (BBI life sciences, #E661006-0200) to each well, and the absorbance values at 450 nm (OD450) was measured using a microplate reader (PerkinElemer, #Enspire).
TABLE-US-00017 TABLE 9-1 Material information of B7 family proteins used in this example Other members of B7 family Supplier Catalog number Human B7-1/CD80 Protein, Fc Tag (HPLC-verified) Acro B71-H5259 Human B7-2/CD86 Protein, Fc Tag Acro CD6-H5257 Human B7-DC/PD-L2/CD273 Protein, Recombinant (Fc Tag) Sino Biological H10292-H02H Human PD-L1/B7-H1 Protein, His Tag (HPLC verified) Acro PD1-H5229 CD275/ICOS ligand Protein, Human, Recombinant (Fc Tag) Sino Biological 11559-H02H B7-H3/CD276 Protein, Human, Recombinant (ECD, Fc Tag) Sino Biological 11188-H02H Recombinant human B7H4 Fc Chimera R&D 8870-B7-050 Recombinant human VISTA/B7H5/PD-1H Fc Chimera R&D 7126-B7-050 B7-H6/NCR3LG1 Protein, Human, Recombinant (Fc Tag) Sino Biological 16140-H02H Recombinant human B7H7/HHLA2 Fc Chimera R&D 8084-B7-050
Example 10. Serum Stability Assay
[0219] 30 μL of antibody was diluted to 270 μL of normal human serum (serum concentration 90%), the antibody was divided into 5 parts which were incubated at 37° C. for 0 day, 1 day, 4 days, 7 days and 14 days, respectively, and then the antibodies were taken out, quick frozen with liquid nitrogen, and stored at −80° C. The binding of antibodies to B7H4 on SK-BR-3 cells was detected by flow methods.
[0220] SK-BR-3 or CHOK1/H B7H4 cells were digested and resuspended with PBS containing 2% BSA. The cell density was adjusted to 1×10.sup.6 cells/mL. The cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 μL/well, followed by the addition of test antibodies diluted in a 3-fold gradient at a concentration that was 2 times the final concentration, each at 100 μL/well. The cells were incubated at 4° C. for 2 h away from light. Thereafter, the cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% BSA, and centrifuged at 500 g at 4° C. for 5 minutes, and then the supernatant was discarded. Then each well was added with 100 μL of fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-098, diluted in a 1:500 ratio), and the plate was incubated away from light at 4° C. for 60 minutes. The cells in each well were washed twice with 100 μL of pre-cooled PBS containing 2% BSA, and centrifuged at 500 g at 4° C. for 5 minutes, and then the supernatant was discarded. Finally, the cells in each well were resuspended with 200 μL of pre-cooled PBS containing 2% BSA, and the fluorescence signal values were read using an ACEA Novocyte3000 flow cytometer. The results in
Example 11. Pharmacokinetics in C57BL/6 Mice
[0221] 6 female Nu/Nu mice with a weight of 18-22 g were selected and administered via tail vein at a dose of 20 mg/kg; the whole blood of 3 mice in one group was collected prior to the administration and 15 minutes, 24 hours (1 day), 4 days and 10 days after the administration, and the whole blood of 3 mice in the other group was collected prior to the administration and 5 hours, 2 days, 7 days and 14 days after the administration. The whole blood was left to stand for 30 min for coagulation and centrifuged at 2,000 rpm for 5 min at 4° C., and the isolated serum sample was cryopreserved at −80° C. until it was taken for analysis. In this example, the drug concentration in the serum of mice was quantitatively determined by ELISA method. The ELISA Fc end overall detection method was performed by capturing a fusion protein containing human Fc in the serum of mice using a goat anti-human Fc polyclonal antibody coating a 96-well plate, and then adding an HRP-labeled goat anti-human Fc secondary antibody. The plasma concentration data were analyzed using Phoenix WinNonlin software (version 8.2) by non-compartmental analysis (NCA) to evaluate the pharmacokinetics.
[0222] Table 11-1 shows the pharmacokinetic parameters of PR002418, PR002421 and control antibody 2 (PR002962). The results in
TABLE-US-00018 TABLE 11-1 Pharmacokinetic parameters of anti-B7H4 antibodies PK parameters PR002421 PR002418 PR002962 T½ (hr) 159.3 157.5 141.5 Vd (mL/kg) 93.3 89.3 93.3 AUCall (μg/mL hr) 36,710 37,786 33,913 Cl (mL/hr/kg) 0.42 0.41 0.48 C0 (μg/mL) 437.9 387.6 440.4
Example 12. Anti-Tumor Efficacy of Anti-B7H4 Antibodies
[0223] 12.1. BALB/c Nude Mouse MDA-MB-468 Tumor Model
[0224] On the day of cell inoculation, each BALB/c nude mouse was inoculated subcutaneously with 1×10.sup.7 tumor cells MDA-MB-468, the cells were resuspended in a PBS/Matrigel (1:1) mixture (0.1 mL/mouse) and inoculated subcutaneously. When the mean tumor volume of each group of mice reached 135 mm.sup.3, 25 mice were divided into 5 groups, with an administration cycle being 2 times per week for 12 administrations via intraperitoneal administration. After the start of administration, the body weight and the tumor volume were measured twice a week. The tumor volume was calculated as follows: tumor volume (mm.sup.3)=0.5×long diameter of tumor×short diameter of tumor.sup.2. The experiment was terminated 39 days after administration and all mice were euthanized.
[0225] The in vivo anti-tumor efficacy of BALB/c nude mouse MDA-MB-468 tumor model is shown in
[0226] 12.2. NSG Mouse MDA-MB-468 Tumor Model with Reconstruction of Human PBMC Immune System
[0227] On the day of cell inoculation, each NCG mouse was inoculated subcutaneously with 5×10.sup.6 tumor cells MDA-MB-468, the cells were resuspended in a PBS/Matrigel (1:1) mixture (0.1 mL/mouse) and inoculated subcutaneously. When the mean tumor volume of each group of mice reached 126 mm.sup.3, 30 mice were divided into 5 groups, each mouse was inoculated intravenously with 5×10.sup.6 human PBMCs, and the cells were resuspended in 200 μL of PBS. The administration was started the next day with an administration cycle of twice a week for 8 administrations via intraperitoneal administration. After the start of administration, the body weight and the tumor volume were measured twice a week. The tumor volume was calculated as follows: tumor volume (mm.sup.3)=0.5×long diameter of tumor×short diameter of tumor.sup.2. The experimental observation was terminated 36 days after administration and then all mice were euthanized.
[0228] The in vivo anti-tumor efficacy of the NSG mouse MDA-MB-468 tumor model with reconstitution of human PBMC immune system is shown in
Example 13. Immunohistochemical Staining (IHC)
[0229] The pathological tissue chip was purchased from Guilin Fanpu Biotech, Inc. It comprises a BRC1021 breast cancer tissue chip, an EMC1021 endometrial cancer tissue chip, an OVC1021 ovarian cancer tissue chip and an MNO1021 normal tissue chip. Paraffin sections had a thickness of 4 μm and were taken with positive control tissue. Dewaxing and washing were performed; antigen retrieval was performed as follows: citric acid at pH 6 was added, the mixture was heated at 125° C. for 5 minutes, sealed for 10 minutes, and cooled at room temperature for 30 minutes; the mixture was washed with water and then with 0.3% hydrogen peroxide for 5 minutes, and then washed with TBST for 3 times for 5 minutes; Dako blocking buffer was used directly, and blocked in an incubation box at room temperature for 20 minutes; the blocking buffer was removed, the primary antibody was added, the antibody diluent Dako was used directly, the mixture was incubated for 60 minutes in an incubation box at room temperature, and a control was substituted with Rabbit IgG; the mixture was washed with TBST for three times, five minutes each time; a secondary antibody, Anti-Rabbit (EnVision+System-HRP Labelled Polymer) was incubated in the incubation box for 30 minutes at room temperature; the mixture was washed with TBST for three times, five minutes each time; after DAB color development, 0.85 mL of distilled water was added into reagents according to the sequence of the reagents A to B to C in an amount of 50 μL, and the mixture was incubated for 5 minutes in an incubation box at room temperature, then washed with distilled water, and counterstained with hematoxylin. The chip was observed under a microscope followed by sealing and reading.
[0230] The results in
Example 14. Structure and Design of B7H4×CD3 Bispecific Antibodies
[0231] Selected anti-B7H4 and anti-CD3 antibodies were used to prepare a bispecific antibody. The prepared B7H4×CD3 bispecific antibody can bind to two targets simultaneously, with one terminus being capable of recognizing B7H4 specifically expressed on tumor cell surfaces and the other terminus being capable of binding to CD3 molecules on T cells. After binding to the surface of a tumor cell, the B7H4×CD3 bispecific antibody molecule can recruit and activate T cells in the vicinity of the tumor cell, thereby killing the tumor cell.
[0232] A and B of
[0233] E and F, G and H, and I and J show B7H4×CD3 bispecific antibody molecules having “2+1” asymmetric structures. For the “2+1” asymmetric structural molecules, structures (5) and (6) involve four protein chains, which comprise the heavy and light chains of the corresponding anti-B7H4 antibody, and the heavy and light chains of the anti-CD3 antibody described above (see
[0234] To minimize the formation of byproducts with mismatched heavy chains (e.g., mismatching of two heavy chains of the anti-CD3 antibody), a mutant heterodimeric Fc region carrying a “knob-hole” mutation and a modified disulfide bond was used, as described in WO2009080251 and WO2009080252. The B7H4×CD3 bispecific antibody has an Fc of IgG1 and carries L234A, L235A and P329G (numbered according to the EU index) mutations on CH3 of the Fc. Each bispecific antibody was generated by co-transfecting simultaneously three or four different mammalian expression vectors encoding: 1) the heavy chain of the corresponding anti-B7H4 antibody, which carries a “Hole” mutation in the Fc region so as to produce a heterodimeric antibody, CH3 of the Fc carrying L234A, L235A and P329G mutations; 2) the heavy chain of the corresponding anti-CD3 antibody, which carries a “knob” mutation in the Fc region so as to produce a heterodimeric antibody, CH3 of the Fc carrying L234A, L235A and P329G mutations; 3) the light chain of the corresponding anti-CD3 antibody. 4) the light chain of the corresponding anti-B7H4 antibody. The “knob” mutation in the Fc region of human IgG1 consists of: T366W, and the “Hole” mutation consists of: T366S, L368A, and Y407V. In addition, S354C in the “knob” Fc region and “Hole” Y349C may be included; they form a pair of disulfide bonds to increase the stability and the yield of the heterodimeric antibody.
[0235] The B7H4×CD3 bispecific antibody molecules constructed in this example are listed in Tables 14-1, 14-2, and 14-3, with the structure numbers in the tables corresponding to
TABLE-US-00019 TABLE 14-1 B7H4 × CD3 bispecific antibody molecules having “1 + 1” Fab-Fc-scFv asymmetric structures Linker peptide Bispecific (between Fc type of Fc type of Structure antibody CD3 B7H4 Structure of Structure of VL_B and CD3 end B7H4 end No. molecules antibody antibody CD3 end B7H4 end VH_B) (mutation) (mutation) 1 PR002849 PR000627 PR001476 scFv(VL-VH) Fab GS_15 Human IgG1 Human IgG1 (knob, AAG) (hole, AAG) 1 PR002850 PR000627 PR002405 scFv(VL-VH) Fab GS_15 Human IgG1 Human IgG1 (knob, AAG) (hole, AAG) 1 PR002851 PR000627 PR002408 scFv(VL-VH) Fab GS_15 Human IgG1 Human IgG1 (knob, AAG) (hole, AAG) 1 PR002852 PR000627 PR002410 scFv(VL-VH) Fab GS_15 Human IgG1 Human IgG1 (knob, AAG) (hole, AAG) 1 PR003733 PR001848 PR002037 Fab scFv(VL-VH) GS_20 Human IgG1 Human IgG1 (knob, LALA) (hole, LALA) 1 PR003899 PR003886 PR002037 Fab scFv(VL-VH) GS_20 Human IgG1 Human IgG1 (knob, LALA) (hole, LALA)
TABLE-US-00020 TABLE 14-2 B7H4 × CD3 bispecific antibody molecules having “1 + 1” Fab-Fc-crossFab asymmetric structures Bispecific Fc type of Fc type of Structure antibody CD3 B7H4 Structure of Structure of CD3 end B7H4 end No. molecules antibody antibody CD3 end B7H4 end (mutation) (mutation) 4 PR002855 PR001924 PR001476 Fab(cross Fab Human IgG1 Human IgG1 Fd/LC) (knob, AAG) (hole, AAG) 4 PR002856 PR001924 PR002405 Fab(cross Fab Human IgG1 Human IgG1 Fd/LC) (knob, AAG) (hole, AAG) 4 PR002857 PR001924 PR002408 Fab(cross Fab Human IgG1 Human IgG1 Fd/LC) (knob, AAG) (hole, AAG) 4 PR002858 PR001924 PR002410 Fab(cross Fab Human IgG1 Human IgG1 Fd/LC) (knob, AAG) (hole, AAG)
TABLE-US-00021 TABLE 14-3 B7H4 × CD3 bispecific antibody molecules having “2 + 1” asymmetric structures Code of B7H4 end Bispecific in structure Fc type of Fc type of Structure antibody CD3 B7H4 Structure of Structure of illustration CD3 end B7H4 end No. molecules antibody antibody CD3 end B7H4 end (A/B) (mutation) (mutation) 6 PR002994 PR001924 PR001476 Fab Fab B Human Human IgG1 IgG1 (knob, (hole, AAG) AAG) 5 PR002987 PR001924 PR001476 Fab(cross Fab B Human Human Fd/LC) IgG1 IgG1 (knob, (hole, AAG) AAG) 7 PR003001 PR000627 PR001476 scFv(VL-VH) Fab B Human Human IgG1 IgG1 (knob, (hole, AAG) AAG) 9 PR003008 PR000627 PR001476 scFv(VL-VH) Fab B Human Human IgG1 IgG1 (knob, (hole, LALA) LALA)
TABLE-US-00022 TABLE 14-4 Linker peptide sequences Name of linker peptide Length Sequence GS_5 5 GGGGS SEQ ID NO: 133 GS_15 15 GGGQSGQGGSGGGGS SEQ ID NO: 134 GS_20 20 GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 135
TABLE-US-00023 TABLE 14-5 Sequence numbers of CD3 antibodies of this example Antibody Light Heavy No. chain chain VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 PR000627 131 86 76 46 53 63 8 20 34 PR001924 111 97 86 78 46 53 63 10 20 34 PR001848 110 96 86 78 46 53 63 10 20 34 PR003886 110 107 86 84 46 53 63 10 20 34
TABLE-US-00024 TABLE 14-6 Sequence numbers of B7H4 × CD3 bispecific antibody molecules of this example Polypeptide Polypeptide Polypeptide Polypeptide Antibody No. chain-1 chain-2 chain-3 chain-4 PR002849 109 118 119 PR002850 114 120 119 PR002851 114 121 119 PR002852 115 122 119 PR002855 97 123 118 109 PR002856 97 123 120 114 PR002857 97 123 121 114 PR002858 97 123 122 115 PR002987 97 124 118 109 PR002994 97 125 118 109 PR003001 126 118 109 PR003008 127 118 109 PR003733 110 128 129 PR003899 110 130 129
TABLE-US-00025 TABLE 14-7 Sequence numbers of CDRs of antigen-binding domains of B7H4 × CD3 bispecific antibody molecules Antigen- Structure Antibody binding No. No. domain No. LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 1 PR002849 #1 46 53 63 8 20 34 #2 47 54 64 9 21 35 1 PR002850 #1 46 53 63 8 20 34 #2 47 54 67 9 23 35 1 PR002851 #1 46 53 63 8 20 34 #2 47 54 67 9 24 35 1 PR002852 #1 46 53 63 8 20 34 #2 48 54 65 11 22 36 1 PR003733 #1 46 53 63 10 20 34 #2 48 54 65 11 22 36 1 PR003899 #1 46 53 63 10 20 34 #2 48 54 65 11 22 36 4 PR002855 #1 46 53 63 10 20 34 #2 47 54 64 9 21 35 4 PR002856 #1 46 53 63 10 20 34 #2 47 54 67 9 23 35 4 PR002857 #1 46 53 63 10 20 34 #2 47 54 67 9 24 35 4 PR002858 #1 46 53 63 10 20 34 #2 48 54 65 11 22 36 5 PR002987 #1 46 53 63 10 20 34 #2 47 54 64 9 21 35 6 PR002994 #1 46 53 63 10 20 34 #2 47 54 64 9 21 35 7 PR003001 #1 46 53 63 8 20 34 #2 47 54 64 9 21 35 9 PR003008 #1 46 53 63 8 20 34 #2 47 54 64 9 21 35
TABLE-US-00026 TABLE 14-8 Expression of B7H4 × CD3 bispecific antibody molecule proteins Yield SEC- Bispecific Expression after first HPLC Structure antibody system and purification purity No. molecules volume (mg/L) (%) 1 PR002849 HEK293F (30 mL) 32.5 84.69 1 PR002850 HEK293F (30 mL) 19.7 84.1 1 PR002851 HEK293F (30 mL) 23.1 83.56 1 PR002852 HEK293F (30 mL) 20.7 86.73 1 PR003733 ExpiCHO (1000 mL) 361 86.33 1 PR003899 ExpiCHO (1000 mL) 375.4 80.94 4 PR002855 HEK293F (30 mL) 52.2 73.08 4 PR002856 HEK293F (30 mL) 28.8 72.03 4 PR002857 HEK293F (30 mL) 27.2 78.06 4 PR002858 HEK293F (30 mL) 57.5 91.2 6 PR002994 HEK293F (30 mL) 20.1 51.76 5 PR002987 HEK293F (30 mL) 17.7 81.48 7 PR003001 HEK293F (30 mL) 21 90.49 9 PR003008 HEK293F (30 mL) 17 86.17
Example 15. Detection of Binding Ability of B7H4×CD3 Bispecific Antibodies to B7H4 by FACS
[0236] SK-BR-3 cells were digested. T cells were isolated using the human T cell isolation kit (Miltenyi, #130-096-535) as described in the method of the instruction, and resuspended with PBS containing 2% BSA. The cell density was adjusted to 1×10.sup.6 cells/mL. The cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 μL/well, followed by the addition of test antibodies diluted in a 3-fold gradient at a concentration that was 2 times the final concentration, each at 100 μL/well. The cells were incubated at 4° C. for 2 h away from light. Thereafter, the cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% BSA, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Then each well was added with 100 μL of fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-098, diluted in a 1:500 ratio), and the plate was incubated away from light at 4° C. for 1 h. The cells in each well were washed twice with 100 μL of pre-cooled PBS containing 2% BSA, and centrifuged at 500 g at 4° C. for 5 minutes, and then the supernatant was discarded. Finally, the cells in each well were resuspended with 200 μL of pre-cooled PBS containing 2% BSA, and the fluorescence signal values were read using an ACEA Novocyte3000 flow cytometer.
[0237] The results of the B7H4×CD3 bispecific antibody molecules having the “1+1” asymmetric structures binding to SK-BR-3 cells are shown in
Example 16. T Cell Killing Experiment
[0238] In this experiment, human primary T cells were used as effector cells, and a cell line SK-BR-3 or MDA-MB-468 highly expressing B7H4, a cell line HCC-1954 moderately expressing B7H4, or a B7H4 negative cell line MDA-MB-231 was used as target cells. The killing efficiency was reflected by the conductivity of the target cell measured using an RTCA instrument from ACEA. A 96-well plate E-plate was first equilibrated with 50 μL of complete medium. Target cells were digested, resuspended in RPM1640 complete medium containing 10% fetal bovine serum and diluted to 4×10.sup.5/mL. The cell suspension was plated on the 96-well E-plate at 50 μL/well, i.e., 2×10.sup.4/well, and incubated overnight at 37° C. The next day, primary T cells were isolated using the T cell isolation kit (Miltenyi, #130-096-535) according to the method of the instruction. 50 μL of fresh culture medium containing 2×10.sup.5 T cells was added to each well, followed by the addition of 50 μL of antibodies diluted in a 4× concentration gradient with a maximum final concentration of 10 nM. A total of 8 concentrations were set in duplicate for each antibody. The conductivity of the target cells was measured in real time. The value at hour 24 was used to calculate the target cell killing efficiency=(1−sample/blank control)×100%. The supernatant was collected after 24 hours and the concentration of IFN-γ was detected by ELISA method. The instructions of IFN gamma Human Uncoated ELISA Kit (Thermo, #88-7316-77) were referred to for the ELISA method.
[0239] The results of the B7H4×CD3 bispecific antibody molecules having the “1+1” asymmetric structures activating T cells and killing target cells SK-BR-3 are shown in
Example 17. NSG Mouse Tumor Model with Reconstruction of Human PBMC Immune System
[0240] On the day of cell inoculation, each NCG mouse was inoculated subcutaneously with 5×10.sup.6 tumor cells MDA-MB-468, the cells were resuspended in a PBS/Matrigel (1:1) mixture (0.1 mL/mouse) and inoculated subcutaneously. When the mean tumor volume of each group of mice reached 126 mm.sup.3, 18 mice were divided into 3 groups, each mouse was inoculated intravenously with 5×10.sup.6 human PBMCs, and the cells were resuspended with 200 μL of PBS. The administration was started the next day with an administration cycle of once a week for a total of 3 administrations via intravenous administration. After the start of administration, the body weight and the tumor volume were measured twice a week. The tumor volume was calculated as follows: tumor volume (mm.sup.3)=0.5×long diameter of tumor×short diameter of tumor.sup.2. The experimental observation was terminated 36 days after administration and then all mice were euthanized.
[0241] The mean tumor volume of the mice in the vehicle control group at day 36 after administration was 942 mm.sup.3. The mean tumor volume of the test drug PR003733 (2 mg/kg) treatment group at day 36 after administration was 590 mm.sup.3, showing a significant difference (p value was 0.048) from that of the vehicle control group, with the tumor growth inhibition rate TGI (%) being 37.31%. The mean tumor volume of the test drug PR003899 (2 mg/kg) treatment group at day 36 after administration was 0 mm.sup.3, with complete regression of tumor, showing a significant difference (p value was 0.0001) from that of the vehicle control group, with the tumor growth inhibition rate TGI (%) being 100% (see
[0242] In the HCC-1954 model, on the day of cell inoculation, each NCG mouse was inoculated subcutaneously with 5×10.sup.6 tumor cells HCC-1954, the cells were resuspended in a PBS/Matrigel (1:1) mixture (0.1 mL/mouse) and inoculated subcutaneously. When the mean tumor volume of each group of mice reached 102 mm.sup.3, 15 mice were divided into 3 groups, each mouse was inoculated intravenously with 3×10.sup.6 human PBMCs, and the cells were resuspended with 200 μL of PBS. The administration was started the next day with a dosing period of once a week for a total of 2 administrations, via intravenous administration. After the start of administration, the body weight and the tumor volume were measured twice a week. The tumor volume was calculated as follows: tumor volume (mm.sup.3)=0.5×long diameter of tumor×short diameter of tumor.sup.2. The experiment was terminated 16 days after administration for observation and then all mice were euthanized.
[0243] The mean tumor volume of the mice in the vehicle control group at day 16 after administration was 622 mm.sup.3. The mean tumor volume of the test drug PR003733 (0.5 mg/kg) treatment group at day 16 after administration was 450 mm.sup.3, showing no significant difference (p value was 0.1) from that of the vehicle control group, with the tumor growth inhibition rate TGI (%) being 27.64%. The mean tumor volume of the test drug PR003899 (0.5 mg/kg) treatment group at day 16 after administration was 322 mm.sup.3, showing a significant difference (p value was 0.0028) from that of the vehicle control group, with the tumor growth inhibition rate TGI (%) being 48.27% (see
[0244] Pharmacodynamic experiments of the two tumor models showed that PR003899 had better efficacy than PR003733.