EGFRVIII ANTIBODY AND CONJUGATE, AND PREPARATION METHOD AND USE THEREOF
20210032351 ยท 2021-02-04
Assignee
Inventors
- Ying Zhang (Shanghai, CN)
- Cuiqing Yang (Shanghai, CN)
- Siqi LIU (Shanghai, CN)
- Yu Zhang (Shanghai, CN)
- Lina Wang (Shanghai, CN)
- Lile Liu (Shanghai, CN)
Cpc classification
A61K47/6889
HUMAN NECESSITIES
C07K16/2863
CHEMISTRY; METALLURGY
C07K2317/51
CHEMISTRY; METALLURGY
A61K47/6851
HUMAN NECESSITIES
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
A61K47/6803
HUMAN NECESSITIES
G01N33/57492
PHYSICS
C07K2317/34
CHEMISTRY; METALLURGY
A61K47/6849
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
Disclosed are an EGFRvIII antibody and a conjugate, and a preparation method and the use thereof. The antibody comprises complementarity-determining regions (CDRs) of the EGFRvIII antibody: one or more of heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and/or one or more of light chain CDR1, light chain CDR2 and light chain CDR3, with the amino acid sequence thereof being as described in the present invention. The EGFRvIII antibody has a high affinity with the EGFRvIII protein, and can enter cells after coupling with small molecule toxins such as MMAE and has a cytotoxic killing effect on EGFRvIII positive cells. The antibody therefore can be used for preparing drugs for treating tumors and other diseases.
Claims
1. An EGFRvIII antibody, wherein the antibody comprises complementarity determining regions (CDRs): one or more of heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and/or one or more of light chain CDR1, light chain CDR2 and light chain CDR3, wherein the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 2, SEQ ID No. 10, SEQ ID No. 18, SEQ ID No. 26, SEQ ID No. 34, SEQ ID No. 42, SEQ ID No. 50, SEQ ID No. 58, SEQ ID No. 66, SEQ ID No. 74, SEQ ID No. 82, SEQ ID No. 90 or SEQ ID No. 98 in the sequence listing; the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 19, SEQ ID No. 27, SEQ ID No. 35, SEQ ID No. 43, SEQ ID No. 51, SEQ ID No. 59, SEQ ID No. 67, SEQ ID No. 75, SEQ ID No. 83, SEQ ID No. 91, SEQ ID No. 99, SEQ ID NO. 184 or SEQ ID NO. 186 in the sequence listing; the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 20, SEQ ID No. 28, SEQ ID No. 36, SEQ ID No. 44, SEQ ID No. 52, SEQ ID No. 60, SEQ ID No. 68, SEQ ID No. 76, SEQ ID No. 84, SEQ ID No. 92 or SEQ ID No. 100 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 6, SEQ ID No. 14, SEQ ID No. 22, SEQ ID No. 30, SEQ ID No. 38, SEQ ID No. 46, SEQ ID No. 54, SEQ ID No. 62, SEQ ID No. 70, SEQ ID No. 78, SEQ ID No. 86, SEQ ID No. 94 or SEQ ID No. 102 in the sequence listing; the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 7, SEQ ID No. 15, SEQ ID No. 23, SEQ ID No. 31, SEQ ID No. 39, SEQ ID No. 47, SEQ ID No. 55, SEQ ID No. 63, SEQ ID No. 71, SEQ ID No. 79, SEQ ID No. 87, SEQ ID No. 95 or SEQ ID No. 103 in the sequence listing; the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 8, SEQ ID No. 16, SEQ ID No. 24, SEQ ID No. 32, SEQ ID No. 40, SEQ ID No. 48, SEQ ID No. 56, SEQ ID No. 64, SEQ ID No. 72, SEQ ID No. 80, SEQ ID No. 88, SEQ ID No. 96 or SEQ ID No. 104 in the sequence listing; or the amino acid sequence of the heavy chain CDR1 is as shown in an amino acid sequence which has at least 80% sequence homology with the amino acid sequence as shown in SEQ ID No. 2, SEQ ID No. 10, SEQ ID No. 18, SEQ ID No. 26, SEQ ID No. 34, SEQ ID No. 42, SEQ ID No. 50, SEQ ID No. 58, SEQ ID No. 66, SEQ ID No. 74, SEQ ID No. 82, SEQ ID No. 90 or SEQ ID No. 98 in the sequence listing; the amino acid sequence of the heavy chain CDR2 is as shown in an amino acid sequence which has at least 80% sequence homology with the amino acid sequence as shown in SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 19, SEQ ID No. 27, SEQ ID No. 35, SEQ ID No. 43, SEQ ID No. 51, SEQ ID No. 59, SEQ ID No. 67, SEQ ID No. 75, SEQ ID No. 83, SEQ ID No. 91, SEQ ID No. 99, SEQ ID NO. 184 or SEQ ID NO. 186 in the sequence listing; the amino acid sequence of the heavy chain CDR3 is as shown in an amino acid sequence which has at least 80% sequence homology with the amino acid sequence as shown in SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 20, SEQ ID No. 28, SEQ ID No. 36, SEQ ID No. 44, SEQ ID No. 52, SEQ ID No. 60, SEQ ID No. 68, SEQ ID No. 76, SEQ ID No. 84, SEQ ID No. 92 or SEQ ID No. 100 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in an amino acid sequence which has at least 80% sequence homology with the amino acid sequence as shown in SEQ ID No. 6, SEQ ID No. 14, SEQ ID No. 22, SEQ ID No. 30, SEQ ID No. 38, SEQ ID No. 46, SEQ ID No. 54, SEQ ID No. 62, SEQ ID No. 70, SEQ ID No. 78, SEQ ID No. 86, SEQ ID No. 94 or SEQ ID No. 102 in the sequence listing; the amino acid sequence of the light chain CDR2 is as shown in an amino acid sequence which has at least 80% sequence homology with the amino acid sequence as shown in SEQ ID No. 7, SEQ ID No. 15, SEQ ID No. 23, SEQ ID No. 31, SEQ ID No. 39, SEQ ID No. 47, SEQ ID No. 55, SEQ ID No. 63, SEQ ID No. 71, SEQ ID No. 79, SEQ ID No. 87, SEQ ID No. 95 or SEQ ID No. 103 in the sequence listing; the amino acid sequence of the light chain CDR3 is as shown in an amino acid sequence which has at least 80% sequence homology with the amino acid sequence as shown in SEQ ID No. 8, SEQ ID No. 16, SEQ ID No. 24, SEQ ID No. 32, SEQ ID No. 40, SEQ ID No. 48, SEQ ID No. 56, SEQ ID No. 64, SEQ ID No. 72, SEQ ID No. 80, SEQ ID No. 88, SEQ ID No. 96 or SEQ ID No. 104 in the sequence listing.
2. The EGFRvIII antibody as defined in claim 1, wherein the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 2 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 3 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 4 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 10 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 11 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 12 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 18 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 19 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 20 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 26 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 27 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 28 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 34 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 35 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 36 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 42 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 43 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 44 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 50 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 51 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 52 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 58 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 59 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 60 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 66 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 67 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 68 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 74 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 75 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 76 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 82 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 83 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 84 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 90 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 91 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 92 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 98 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 99 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 100 in the sequence listing; the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 2 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 184 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 4 in the sequence listing; or the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 2 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 186 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 4 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 6 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 7 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 8 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 14 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 15 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 16 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 22 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 23 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 24 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 30 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 31 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 32 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 38 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 39 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 40 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 46 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 47 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 48 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 54 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 55 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 56 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 62 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 63 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 64 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 70 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 71 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 72 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 78 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 79 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 80 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 86 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 87 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 88 in the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 94 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 95 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 96 in the sequence listing; or the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 102 in the sequence listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 103 in the sequence listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 104 in the sequence listing.
3. The EGFRvIII antibody as defined in claim 1, wherein the antibody further comprises an antibody framework region containing a heavy chain framework region and/or a light chain framework region; preferably, the heavy chain framework region is a human or murine antibody heavy chain framework region, and/or the light chain framework region is a human or murine antibody light chain framework region; more preferably, the light chain framework region is a human antibody light chain framework region, preferably a combination of human antibody light chain framework segments of FR1, FR2 and FR3 of IGKV1-39*01 or IGKV3-11*01 and segment FR4 of J.sub.K-2 or J.sub.K-4 of a human germline antibody light chain; and the heavy chain framework region is a human antibody heavy chain framework region, preferably a combination of human antibody heavy chain framework segments of FR1, FR2 and FR3 of IGHV1-46*01 and FR4 of J.sub.H-4 or J.sub.H-6b of a human germline antibody heavy chain.
4. The EGFRvIII antibody as defined in claim 3, wherein the EGFRvIII antibody comprises a heavy chain variable region and/or light chain variable region containing the CDRs, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 33, SEQ ID No. 41, SEQ ID No. 49, SEQ ID No. 57, SEQ ID No. 65, SEQ ID No. 73, SEQ ID No. 81, SEQ ID No. 89, SEQ ID No. 97, SEQ ID No. 179, SEQ ID No. 180, SEQ ID NO. 133, SEQ ID NO. 134, SEQ ID NO. 135, SEQ ID NO. 136, SEQ ID NO. 141, SEQ ID NO. 142, SEQ ID NO. 143, SEQ ID NO. 144, SEQ ID NO. 145, SEQ ID NO. 146 or SEQ ID NO. 147 in the sequence listing; the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 5, SEQ ID No. 13, SEQ ID No. 21, SEQ ID No. 29, SEQ ID No. 37, SEQ ID No. 45, SEQ ID No. 53, SEQ ID No. 61, SEQ ID No. 69, SEQ ID No. 77, SEQ ID No. 85, SEQ ID No. 93, SEQ ID No. 101, SEQ ID NO. 137, SEQ ID NO. 138, SEQ ID NO. 139, SEQ ID NO. 140, SEQ ID NO. 148, SEQ ID NO. 149, SEQ ID NO. 150, SEQ ID NO. 151, SEQ ID NO. 152 or SEQ ID NO. 153 in the sequence listing; or the amino acid sequence of the heavy chain variable region is as shown in an amino acid sequence which has at least 80% sequence homology with the amino acid sequence as shown in SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 33, SEQ ID No. 41, SEQ ID No. 49, SEQ ID No. 57, SEQ ID No. 65, SEQ ID No. 73, SEQ ID No. 81, SEQ ID No. 89, SEQ ID No. 97, SEQ ID No. 179, SEQ ID No. 180, SEQ ID NO. 133, SEQ ID NO. 134, SEQ ID NO. 135, SEQ ID NO. 136, SEQ ID NO. 141, SEQ ID NO. 142, SEQ ID NO. 143, SEQ ID NO. 144, SEQ ID NO. 145, SEQ ID NO. 146 or SEQ ID NO. 147 in the sequence listing; the sequence of the light chain variable region is as shown in an amino acid sequence which has at least 80% sequence homology with the amino acid sequence as shown in SEQ ID No. 5, SEQ ID No. 13, SEQ ID No. 21, SEQ ID No. 29, SEQ ID No. 37, SEQ ID No. 45, SEQ ID No. 53, SEQ ID No. 61, SEQ ID No. 69, SEQ ID No. 77, SEQ ID No. 85, SEQ ID No. 93, SEQ ID No. 101, SEQ ID NO. 137, SEQ ID NO. 138, SEQ ID NO. 139, SEQ ID NO. 140, SEQ ID NO. 148, SEQ ID NO. 149, SEQ ID NO. 150, SEQ ID NO. 151, SEQ ID NO. 152 or SEQ ID NO. 153 in the sequence listing.
5. The EGFRvIII antibody as defined in claim 4, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 1 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 5 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 9 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 13 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 17 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 21 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 25 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 29 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 33 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 37 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 41 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 45 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 49 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 53 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 57 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 61 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 65 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 69 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 73 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 77 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 81 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 85 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 89 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 93 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 97 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 101 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 179 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 5 in the sequence listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 180 in the sequence listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 5 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 133 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 137 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 133 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 138 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 133 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 139 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 133 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 140 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 134 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 137 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 134 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 138 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 134 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 139 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 134 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 140 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 135 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 137 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 135 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 138 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 135 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 139 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 135 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 140 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 136 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 137 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 136 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 138 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 136 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 139 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 136 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 140 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 143 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 149 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 144 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 149 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 145 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 149 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 146 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 149 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 147 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 149 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 143 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 148 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 143 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 150 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 143 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 151 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 143 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 152 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 143 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 153 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 141 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 149 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 141 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 150 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 141 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 151 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 141 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 152 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 141 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 153 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 142 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 149 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 142 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 150 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 142 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 151 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 142 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 152 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 142 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 153 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 145 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 148 in the sequence listing; the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 146 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 148 in the sequence listing; or the amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID No. 147 in the sequence listing, and the amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID No. 148 in the sequence listing.
6. The EGFRvIII antibody as defined in claim 1, wherein the EGFRvIII antibody further comprises an antibody heavy chain constant region and/or an antibody light chain constant region, wherein the antibody heavy chain constant region is preferably a human or mouse antibody heavy chain constant region, and the antibody light chain constant region is preferably a human or mouse antibody light chain constant region; preferably, the EGFRvIII antibody is a monoclonal antibody, full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single chain antibody fragment, single domain antibody or single-domain antibody of the EGFRvIII.
7. (canceled)
8. A nucleic acid, wherein the nucleic acid encodes the EGFRvIII antibody as defined in claim 1; preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 105, SEQ ID No. 107, SEQ ID No. 109, SEQ ID No. 111, SEQ ID No. 113, SEQ ID No. 115, SEQ ID No. 117, SEQ ID No. 119, SEQ ID No. 121, SEQ ID No. 123, SEQ ID No. 125, SEQ ID No. 127, SEQ ID No. 129, SEQ ID No. 185, SEQ ID No. 154, SEQ ID No. 155, SEQ ID No. 156, SEQ ID No. 157, SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 165, SEQ ID No. 166, SEQ ID No. 167 or SEQ ID No. 168 in the sequence listing; and/or the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 106, SEQ ID No. 108, SEQ ID No. 110, SEQ ID No. 112, SEQ ID No. 114, SEQ ID No. 116, SEQ ID No. 118, SEQ ID No. 120, SEQ ID No. 122, SEQ ID No. 124, SEQ ID No. 126, SEQ ID No. 128, SEQ ID No. 130, SEQ ID No. 158, SEQ ID No. 159, SEQ ID No. 160, SEQ ID No. 161, SEQ ID No. 169, SEQ ID No. 170, SEQ ID No. 171, SEQ ID No. 172, SEQ ID No. 173 or SEQ ID No. 174 in the sequence listing.
9. (canceled)
10. The nucleic acid as defined in claim 8, wherein the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 105 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 106 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 107 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 108 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 109 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 110 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 111 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 112 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 113 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 114 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 115 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 116 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 117 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 118 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 119 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 120 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 121 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 122 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 123 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 124 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 125 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No 126 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 127 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 128 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 129 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 130 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 185 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 106 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 154 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 158 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 154 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 159 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 154 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 160 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 154 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 161 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 155 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 158 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 155 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 159 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 155 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 160 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 155 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 161 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 156 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 158 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 156 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 159 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 156 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 160 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 156 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 161 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 157 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 158 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 157 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 159 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 157 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 160 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 157 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 161 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 164 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 170 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 165 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 170 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 166 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 170 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 167 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 170 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 168 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 170 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 164 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 169 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 164 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 171 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 164 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 172 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 164 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 173 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 164 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 174 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 162 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 170 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 162 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 171 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 162 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 172 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 162 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 173 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 162 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 174 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 163 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 170 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 163 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 171 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 163 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 172 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 163 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 173 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 163 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 174 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 166 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 169 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 167 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 169 in the sequence listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 168 in the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 169 in the sequence listing.
11. A recombinant expression vector comprising the nucleic acid as defined in claim 8.
12. A recombinant expression transformant comprising the recombinant expression vector as defined in claim 11.
13. A method for preparing the EGFRvIII antibody, comprising the following steps: culturing the recombinant expression transformant as defined in claim 12, and obtaining the EGFRvIII antibody from the culture.
14. An immunoconjugate, wherein the immunoconjugate comprises the EGFRvIII antibody as defined in claim 1 covalently attached to a cytotoxic agent.
15. The immunoconjugate as defined in claim 14, wherein 1 equivalent of the EGFRvIII antibody is linked to y equivalent of the cytotoxic agent via x equivalent of a linker, and the immunoconjugate has a structure as shown in formula 1,
Ab-(L).sub.x-(D).sub.yformula 1 where Ab is the EGFRvIII antibody; L is a linker; D is a cytotoxic agent; x is a natural number, preferably 1 to 20; y is a natural number greater than 0, preferably 1 to 20; x and y are each independently and more preferably 2w, with w being an integer of 1 to 5, further preferably 3 and 4; and the ratio of x and y is preferably 1:1.
16. The immunoconjugate as defined in claim 15, wherein the linker L comprises the structure of formula 2, which is the remaining part in L after the leaving group leaves:
(CO-Alk.sup.1-Sp.sup.1-Ar-Sp.sup.2-Alk.sup.2-C(Z.sup.1)=Q-Sp)formula 2 preferably, the linker L is maleimidocaproyl (MC) or maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol (MC-VC-PAB); and/or the D is monomethylauristatin F (MMAF) or monomethylauristatin E (MMAE).
17. The immunoconjugate as defined in claim 14, wherein in the formula 1, x=y=n, and the structure of the immunoconjugate is as shown in formula 3-1 or 3-2, ##STR00003## in the formula 3-1, m is 1 to 10, preferably m is 5, and L is maleimidocaproyl; D is monomethylauristatin F (MMAF); where n is a natural number, preferably an integer of 1 to 20, and more preferably 2w, with w being an integer of 1 to 5, further preferably 3 and 4; ##STR00004## in the formula 3-2, m is 1 to 10, preferably m is 5, and L is maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol; D is monomethylauristatin E (MMAE); where n is a natural number, preferably an integer of 1 to 20, and more preferably 2w, with w being an integer of 1 to 5, further preferably 3 and 4.
18. A pharmaceutical composition, wherein the pharmaceutical composition comprises the EGFRvIII antibody as defined in claim 1, and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition comprises 0.01% to 99.99% of the EGFRvIII antibody as defined in claim 1, and 0.01% to 99.99% of the pharmaceutically acceptable carrier, wherein the percentage is a mass percentage accounting for the pharmaceutical composition.
19. (canceled)
20. A method for treating tumor in a subject in need thereof, comprising administrating an effective amount of the EGFRvIII antibody as defined in claim 1 to the subject.
21. A method for detecting a cell overexpressing an EGFRvIII protein, wherein the method comprises the following steps: contacting the EGFRvIII antibody as defined in claim 1 with a sample to be detected in vitro, then detecting the binding of the EGFRvIII antibody as defined in claim 1 to the sample to be detected.
22. A composition for detecting a cell overexpressing an EGFRvIII protein, wherein the composition comprises the EGFRvIII antibody as defined in claim 1 as an active ingredient.
23. A method for preventing or treating a disease related to abnormal expression or function of the EGFRvIII, comprising administrating an effective amount of the EGFRvIII antibody as defined in claim 1 to the subject; preferably, the disease related to abnormal expression or function of the EGFRvIII is a tumor, wherein the tumor is preferably bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, or renal cancer.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0212] The following examples further illustrate the present invention, but the present invention is not limited thereto. In experimental methods in the following examples where no specific conditions are indicated, choices can be made according to conventional methods and conditions or commodity instructions.
[0213] The following examples further illustrate the present invention, but the present invention is not limited thereto. In experimental methods in the following examples where no specific conditions are indicated, choices can be made according to conventional methods and conditions or commodity instructions.
[0214] The room temperature described in the examples is a conventional room temperature in the art, generally is 10 C. to 30 C.
Example 1 Preparation of EGFRvIII Antibody
(I) Preparation of Immunogen A
[0215] The nucleotide sequences containing the amino acid sequences (NCBI: NP 005219.2, with deletion of amino acids at positions 30-297) encoding the extracellular region of the human EGFRvIII protein are cloned into a pCpC vector (purchased from Invitrogen, V044-50) with a human IgG Fc fragment (hFc); and plasmids are prepared according to established standard molecular biology methods, and for specific methods, please see Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, N.Y.: Cold Spring Harbor Laboratory Press). HEK293 cells (purchased from Invitrogen) are subjected to transient transfection (PEI, Polysciences) and an expanded culture is performed using FreeStyle 293 (Invitrogen) at 37 C. After 4 days, the cell culture broth is collected, and the cell components are removed by centrifugation to obtain a culture supernatant containing the extracellular region of the EGFRvIII protein. The culture supernatant is loaded onto a protein A affinity chromatography column (Mabselect Sure, purchased from GE Healthcare), and the change in ultraviolet absorption values (A280 nm) is monitored with an ultraviolet (UV) detector. After loading, the protein A affinity chromatography column is washed with PBS phosphate buffer (pH 7.2) until the UV absorption value returns back to the baseline, and then eluted with 0.1 M glycine hydrochloric acid (pH 2.5) to collect the hFc-tagged EGFRvIII protein (EGFRvIII-hFc) eluted from the protein A affinity chromatography column, which is dialyzed with the PBS phosphate buffer (pH 7.2) in the refrigerator at 4 C. overnight. The dialyzed protein is sterile filtered through 0.22 microns, and then packaged and stored at 80 C. to obtain a purified immunogen A.
(II) Preparation of Immunogen B
[0216] The nucleotide sequence encoding the full-length amino acid sequence of the human EGFRvIII (NCBI: NP_005219.2, with deletion of amino acids at positions 30-297) and the nucleotide sequence encoding the full-length amino acid sequence of the human EGFR (NCBI: NP_005219.2) are cloned into a pIRES vector (purchased from Clontech), and plasmids are prepared. The HEK293 cell line, U87MG cell line and CHO-K1 cell line (all purchased from Invitrogen) are subjected to plasmid transfection (PEI, purchased from Polysciences), and then selectively cultured for 2 weeks in a DMEM medium containing 0.5 g/ml of 10% (w/w) fetal bovine serum. The culture is subcloned in a 96-well culture plate by using a limiting dilution method, and cultured at 37 C., 5% (v/v) CO.sub.2; after about 2 weeks, a part of monoclonal wells are selected and amplified into a 6-well plate. The amplified clones are screened by a flow cytometry analysis method using a known EGFRvIII antibody (purchased from Absoluteantibody, #Ab00184-1.1). The monoclonal cell line with better growth vigour and higher fluorescence intensity is selected for further expanded culture and same was cryopreserved in liquid nitrogen to obtain an immunogen B. The specific selection results are as shown in table 3 and
TABLE-US-00005 TABLE 3 Results of FACS detection of CHO-K1 stably transfecting cell lines of human EGFRvIII protein Cell mean fluorescence intensity Serial Clone numbers of stably IgG subtype EGFRvIII number transfected cell lines control antibody 1 CHO-K1-hEGFRvIII 1G10 2.4 563.0 2 CHO-K1-hEGFRvIII 1D9 1.9 457.0 3 CHO-K1-hEGFRvIII 2B3 1.9 109.0 4 CHO-K1-hEGFRvIII 1E10 2.0 92.0 5 CHO-K1-hEGFRvIII 2D11 1.8 77.0 6 CHO-K1-hEGFRvIII 1E3 2.4 42.0
TABLE-US-00006 TABLE 4 Results of FACS detection of CHO-K1 stably transfecting cell lines of human EGFR protein Cell mean fluorescence intensity Serial Clone numbers of stably IgG subtype EGFR number transfected cell lines control antibody 1 CHO-K1-hEGFR 3G2 2.3 396.9 2 CHO-K1-hEGFR 3B4 2.1 285.6 3 CHO-K1-hEGFR 1G10 2.2 191.5 4 CHO-K1-hEGFR 2F5 2.0 321.5
TABLE-US-00007 TABLE 5 Results of FACS detection of HEK293F stably transfecting cell lines of human EGFRvIII protein Cell mean fluorescence intensity Serial Clone numbers of stably IgG subtype EGFRvIII number transfected cell lines control antibody 1 293F-hEGFRvIII 1C10 4.3 458.1 2 293F-hEGFRvIII 1F3 4.1 395.0 3 293F-hEGFRvIII 1D11 3.6 388.0
TABLE-US-00008 TABLE 6 Results of FACS detection of U87MG stably transfecting cell lines of human EGFRvIII protein Cell mean fluorescence intensity Serial Clone numbers of stably IgG subtype EGFRvIII number transfected cell lines control antibody 1 U87MG-hEGFRvIII 2G2 1.8 181.9 2 U87MG-hEGFRvIII 2C6 1.8 178.0 3 U87MG-hEGFRvIII 1D1 3.0 96.4 4 U87MG-hEGFRvIII 2C3 1.9 82.7 5 U87MG-hEGFRvIII 1F9 1.7 68.0 6 U87MG-hEGFRvIII 1B3 2.3 66.9
(III) Preparation of Hybridoma Cells and Antibody Screening
[0217] A. 6 to 8 week-old BALB/cAnNCrl mice or SJL/JorllcoCrl mice (purchased from Shanghai SLAC Laboratory Animal Co., Ltd.) are used in the immunization with immunogen A, and the mice are raised under SPF conditions. For the primary immunization, an immunogen A protein is emulsified with a Freund's complete adjuvant and each mouse is intraperitoneally injected at 0.25 ml, namely, each mouse is injected with 50 g of the immunogen A protein. For the booster immunization, an immunogen A protein is emulsified with a Freund's incomplete adjuvant and each mouse is intraperitoneally injected at 0.25 ml, namely, each mouse is injected with 50 g of the immunogen A protein. The interval between the primary immunization and the first booster immunization is 2 weeks, and the interval between subsequent booster immunizations is 3 weeks. Blood is collected 1 week after each booster immunization, and the antibody titer and specificity of immunogen A in the serum are detected by ELISA and FACS, with the results as shown in
TABLE-US-00009 TABLE 7 Serum antibody titer in Balb/c mice immunized with EGFRvIII protein detected by ELISA OD450 nm Serum dilutability Blank Batch 1:100 1:10.sup.3 1:10.sup.4 1:10.sup.5 1:10.sup.6 1:10.sup.7 control 636 (TB2) 2.49 2.82 2.34 0.91 0.37 0.20 0.24 637 (TB2) 2.64 2.63 2.07 0.57 0.20 0.18 0.17 638 (TB2) 2.83 2.89 2.53 1.22 0.27 0.25 0.18 639 (TB2) 2.62 2.65 2.11 0.72 0.22 0.16 0.16 640 (TB2) 2.35 2.59 1.85 0.85 0.26 0.16 0.17
TABLE-US-00010 TABLE 8 Serum antibody titer in SJL mice immunized with EGFRvIII protein detected by ELISA OD450 nm Serum dilutability Blank Batch 1:100 1:10.sup.3 1:10.sup.4 1:10.sup.5 1:10.sup.6 1:10.sup.7 control 641 (TB2) 2.42 2.18 2.31 0.87 0.28 0.15 0.23 642 (TB2) 2.34 2.73 2.33 1.31 0.36 0.23 0.17 643 (TB2) 2.58 2.87 2.46 1.01 0.27 0.18 0.24 644 (TB2) 2.69 2.88 2.11 1.44 0.49 0.19 0.18 645 (TB2) 2.63 2.58 2.60 1.03 0.25 0.17 0.21
[0218] B. 6 to 8 week-old BALB/cAnNCrl mice or SJL/JorllcoCrl mice (purchased from Shanghai SLAC Laboratory Animal Co., Ltd.) are used in the immunization with immunogen B, and the mice are raised under SPF conditions. The HEK293 cell line is transfected with the pIRES plasmid containing the nucleotide sequence encoding the full-length amino acid sequence of human EGFRvIII [see step (II) in example 1] to obtain an HEK293 stable cell line 293F-hEGFRvIII 1C10 containing human EGFRvIII (using X-treme GENE HP DNA Transfection Reagent, which is purchased from Roche, Cat #06 366 236 001, and operating according to the instructions); the cell line is subjected to an expanded culture to 90% confluence in a T-75 cell culture flask; the medium is sucked out, and the cells are washed twice with a DMEM basal medium (purchased from Invitrogen), and then treated with an enzyme-free cell dissociation solution (purchased from Invitrogen) at 37 C. until cells can be fallen off from the wall of the culture dish, and the cells are collected. The cells are washed twice with the DMEM basal medium and are counted, and then the cells are diluted with a phosphate buffer (pH 7.2) to 210.sup.7 cells/ml. Each mouse is injected intraperitoneally with 0.5 ml of the cell suspension at each immunization. The interval between the first immunization and the second immunization is 2 weeks, and the interval between subsequent immunizations is 3 weeks. In addition to the first immunization, blood is collected 1 week after each immunization, and the antibody titer and specificity in the serum are detected by FACS. After the second booster immunization, the serum antibody titer reaches at least 1:1000 detected by FACS.
[0219] Before the completion of steps A and B, each selected mouse is the last time immunized intraperitoneally with 100 g of the purified immunogen A (mice immunized with the immunogen A) or immunogen B (mice immunized with the immunogen B); after 5 days, the mice are sacrificed and spleen cells are collected. NH.sub.4OH is added to a final concentration of 1% (w/w) and red blood cells incorporated in the spleen cells are lysed to obtain a spleen cell suspension. The cells are washed with DMEM basic medium and centrifuged at 1000 rpm for 3 times, then are mixed at a 5:1 ratio of living cell number with mouse myeloma cells SP2/0 (purchased from ATCC), then are subjected to cell fusion by using an efficient electrical fusion method (see METHODS IN ENZYMOLOGY, VOL. 220). The fused cells are diluted into a DMEM medium containing 20% fetal bovine serum and 1HAT, with the percentage being the mass percentage. Then the cells at 110.sup.5/200 L/well are added to a 96-well cell culture plate and placed in an incubator with 5% CO.sub.2 and 37 C., with the percentage being the volume percentage. After 14 days, the cell fusion plate supernatant is screened by ELISA and Acumen (a microplate cell detection method), and the positive clones with OD.sub.450 nm>1.0 in ELISA and MFI value >100 in Acumen are amplified to a 24-well plate, and an expanded culture is performed in DMEM (invitrogen) containing 10% (w/w) HT fetal bovine serum under the conditions of 37 C. and 5% (v/v) CO.sub.2. After culturing for 3 days, the culture broth from the expanded culture in the 24-well plate is centrifuged; the supernatant is collected and analyzed for antibody subtypes; and the binding activity in the EGFRvIII protein and EGFRvIII positive cells (for the detection method of the binding activity, please refer to example 3A and example 3B respectively), and the anti-mouse antibody-MMAF indirect cytotoxic killing experiment (for detection method of the indirect cytotoxic killing activity, please refer to example 4) are determined by ELISA and FACS.
[0220] According to the screening results of the 24-well plate, hybridoma cells with OD.sub.450 nm>1.0 in the ELISA experiment, with the MFI value >50 in the FACS experiment, and with the killing rate of the hybridoma cell culture supernatant for the EGFRvIII-positive cells reaching 50% in the indirect cytotoxicity killing experiment are selected as eligible positive clones, and eligible hybridoma cells are subcloned in the 96-well plate by a limiting dilution method and cultured in a DMEM medium (purchased from invitrogen) containing 10% (w/w) FBS under conditions of 37 C. and 5% (v/v) CO.sub.2. 10 days after subcloning, preliminary screening is performed by ELISA and Acumen, and a single positive monoclone is selected and amplified to a 24-well plate for further cultivation. 3 days later, the FACS is used to determine the positive antigen binding, and the anti-mouse antibody-MMAF indirect cytotoxicity killing experiment is used to evaluate the biological activity (evaluation criteria: OD.sub.450 nm>1.0 in the ELISA experiment, the MFI value >50 in the FACS experiment, and the killing rate of the hybridoma cell culture supernatant for the EGFRvIII-positive cells reaching 50% in the indirect cytotoxicity killing experiment).
[0221] Based on the detection results of the samples in the 24-well plate, the best clones are selected, subjected to an expanded culture in the DMEM medium (purchased from invitrogen) containing 10% (w/w) FBS under conditions of 37 C. and 5% (v/v) CO.sub.2, and then cryopreserved in liquid nitrogen to obtain the hybridoma cells of the present invention, which can be used for subsequent antibody production and purification.
Example 2 Production and Purification of Lead Antibody
[0222] The antibody produced by the hybridoma cells has a relatively low concentration which is only about 1 to 10 g/ml, and the concentration varied greatly. Moreover, various proteins produced by cell culture in the culture medium and fetal bovine serum components contained in the culture medium interfered with many biological activity analysis methods in varying degrees, and therefore, it is necessary to perform a small-scale (1 to 5 mg) antibody production and purification.
[0223] The hybridoma cells obtained in example 1 are inoculated into a T-75 cell culture flask and acclimated and passaged for 3 generations by using a production medium (Hybridoma serum free medium, purchased from Invitrogen). When the cells have a good growing status, they are inoculated into a roller bottle for tissue culture. 200 ml of the production medium is added to each 2 liter of the culture roller bottle, with the cell density inoculated being 1.010.sup.5/ml. The bottle cap is screwed down, and the roller bottle is placed on a rotary machine in a 37 C. incubator at a speed of 3 rpm/min. After a continuous rotating culture for 14 days, the cell culture broth is collected and filtered to remove cells, and the culture supernatant is filtered with a 0.45 micron filter membrane until it is clear. The clear culture supernatant may be purified immediately or cryopreserved at 30 C.
[0224] Monoclonal antibodies in the clear culture supernatant (200 mL) of hybridoma cells are purified by using a 2 mL protein A column (purchased from GE Healthcare). The protein G column is first equilibrated with an equilibration buffer (PBS phosphate buffer, pH 7.4), and then the clear culture supernatant is loaded onto the protein A column, with the flow rate controlled at 3 mL/min. After loading, the protein G column is washed with the equilibration buffer at a volume of 4 column bed volumes of the protein G column. The EGFRvIII antibody bound to the protein A column is eluted with an eluent (0.1 M sodium citrate buffer, pH 3.5), and the elution status is monitored with an ultraviolet detector (A280 ultraviolet absorption peak). The eluted antibodies are collected, and 10% 1.0 M Tris-HCl buffer is added to neutralize the pH, with the percentage being the volume percentage, and then immediately dialyzed with the PBS phosphate buffer overnight. The next day, the dialysate is changed once and is further dialyzed for 3 hours. The dialyzed EGFRvIII antibody is collected, aseptically filtered with a 0.22 micron filter, and stored aseptically to obtain a purified EGFRvIII antibody.
[0225] The purified EGFRvIII antibody is detected and analyzed for protein concentration (A280/1.4), purity, endotoxin (Lonza kit), etc. The results as shown in table 9 indicate that the endotoxin concentration of the final antibody product is within 1.0 EU/mg.
TABLE-US-00011 TABLE 9 Quality control of purified EGFRvIII antibody Protein Antibody Clone Antibody concentration Endotoxin name number purity (mg/ml) (EU/mg) Murine anti- 75G7C6 >95% 0.44 <1.0 75G7 Murine anti- 63A10A7 >95% 1.26 <1.0 63A10 Murine anti- 64F1F8 >95% 1.52 <1.0 64F1 Murine anti- 7E5-2A9 >95% 0.81 <1.0 7E5 Murine anti- 43H6A1 >95% 1.08 <1.0 43H6 Murine anti- 46C4A6 >95% 1.43 <1.0 46C4 Murine anti- 49G12G5 >95% 1.16 <1.0 49G12 Murine anti- 51G7C2 >95% 1.07 <1.0 51G7 Murine anti- 53D8H4 >95% 0.93 <1.0 53D8 Murine anti- 54D2G10 >95% 1.29 <1.0 5402 Murine anti- 56F10G2 >95% 0.92 <1.0 56F10 Murine anti- 59G3F12 >95% 1.29 <1.0 59G3 Murine anti- 64B11F2 >95% 1.11 <1.0 64B11
Example 3 Detection and Identification of Lead Antibody
A. Binding of the Antibody to an EGFRvIII Protein Detected by an Enzyme-Linked Immunosorbent Assay (ELISA)
[0226] The purified EGFRvIII antibody obtained in example 2 is reacted with a human EGFRvIII-hFc protein.
[0227] The purified immunogen A (EGFRvIII-hFc) obtained in example 1 (for the preparation method, see step (I) in example 1) is diluted with PBS to a final concentration of 1.0 g/mL, and then added to a 96-well ELISA plate at 100 l/well. The plate is sealed with a plastic film and incubated at 4 C. overnight. The next day, the plate is washed twice with a plate washing solution [PBS+0.01% (v/v) Tween20], and added with a blocking solution [PBS+0.01% (v/v) Tween20+1% (w/w) BSA] for blocking 2 hours at room temperature. The blocking solution is discarded, and the purified EGFRvIII antibody obtained in example 2 is added at 100 l/well. The plate is incubated for 2 hours at 37 C., and then washed 3 times with the plate washing solution [PBS+0.01% (v/v) Tween20]. The plate is added with an HRP (horseradish peroxidase) labeled secondary antibody (purchased from Sigma), incubated at 37 C. for 2 hours, and then washed 3 times with the plate washing solution [PBS+0.01% (v/v) Tween20]. The plate is added with 100 l of TMB substrate/well, incubated for 30 minutes at room temperature, and then added with 100 l of stopping solution (1.0N HCl)/well. A450 nm value is read using an ELISA plate reader (SpectraMax 384plus, purchased from Molecular Device), and the results are shown in
TABLE-US-00012 TABLE 10 Binding reaction of EGFRvIII antibody with human EGFRvIII-hFc protein detected by ELISA OD450 nm Antibody Clone Antibody concentration (nM) name number 100 10 1 0.1 0.01 0.001 0.0001 Blank Murine anti- 75G7C6 0.56 0.19 0.09 0.07 0.06 0.07 0.07 0.07 75G7 Murine anti- 63A10A7 3.71 3.76 3.42 0.80 0.17 0.09 0.08 0.08 63A10 Murine anti- 64F1F8 3.73 3.76 3.51 1.00 0.16 0.09 0.08 0.08 64F1 Murine anti- 7E5-2A9 3.36 3.14 3.18 1.68 0.35 0.17 0.15 0.19 7E5 Murine anti- 43H6A1 3.85 3.87 3.86 1.39 0.25 0.11 0.09 0.10 43H6 Murine anti- 46C4A6 3.83 3.85 3.50 0.72 0.15 0.09 0.08 0.08 46C4 Murine anti- 49G12G5 3.79 3.80 3.20 0.64 0.14 0.13 0.08 0.08 49G12 Murine anti- 51G7C2 3.68 3.69 2.44 0.46 0.15 0.09 0.08 0.09 51G7 Murine anti- 53D8H4 3.72 3.69 3.20 0.64 0.16 0.10 0.09 0.09 53D8 Murine anti- 54D2G10 3.61 3.60 1.33 0.22 0.09 0.08 0.08 0.08 54D2 Murine anti- 56F10G2 3.61 3.53 0.98 0.18 0.11 0.10 0.09 0.11 56F10 Murine anti- 59G3F12 3.71 3.67 2.68 0.49 0.13 0.08 0.08 0.08 59G3 Murine anti- 64B11F2 3.64 3.63 1.58 0.24 0.09 0.08 0.08 0.08 64B11 hIgG IgG control 0.11 0.10 0.09 0.09 0.09 0.09 0.09 0.09
B. Binding of Antibody to EGFRvIII Expressing Cell Detected by Fluorescence Activated Cell Sorting (FACS)
[0228] The desired cells are subjected to an expanded culture to 90% confluence in a T-75 cell culture flask; the medium is sucked out, and the cells are washed twice with a HBSS buffer (Hanks Balanced Salt Solution) (purchased from Invitrogen), and then treated with an enzyme-free cell dissociation solution (Versene solution: purchased from Life Technology) and collected. The cells are washed twice with the HBSS buffer and are counted, and then the cells are diluted with the HBSS buffer to 210.sup.6 cells/ml, added with 1% goat serum blocking solution, with the percentage being the mass percentage, and incubated on ice for 30 minutes, and then centrifugally washed twice with the HBSS buffer. The collected cells are suspended to 210.sup.6 cells/mL with an FACS buffer (HBSS+1% BSA, with the percentage being the mass percentage), added to a 96-well FACS reaction plate at 100 L/well, then added with 100 L/well of the purified EGFRvIII antibody (samples to be tested) obtained in example 2, and incubated on ice for 2 hours. The plate is centrifugally washed twice with the FACS buffer, added with 100 L/well of a fluorescence (Alexa 488) labeled secondary antibody (purchased from Invitrogen), and then incubated on ice for 1 hour. The plate is centrifugally washed 3 times with the FACS buffer, added with 100 L/well of a fixing solution [4% (v/v) paraformaldehyde] for suspending the cells, and centrifugally washed twice with the FACS buffer after 10 minutes. The cells are suspended in 100 L of the FACS buffer, and the results are detected and analyzed by FACS (FACS Calibur, purchased from BD). Data analysis is performed by using the software (CellQuest) to obtain the mean fluorescence intensity (MFI) of the cells. Data is further analyzed by using the software (GraphPad Prism5) to perform data fitting and calculate EC50. The analysis results as shown in table 11 and
TABLE-US-00013 TABLE 11 Binding reaction of EGFRvIII antibody with U87MG-hEGFRvIII, A431, U87MG and HEB cells detected by FACS Antibody EC50, nM Antibody clone U87MG- name number hEGFRvIII A431 HEB U87MG Murine anti- 75G7C6 4.8 Weakly positive Negative Negative 75G7 Murine anti- 63A10A7 3.1 Weakly positive Negative Negative 63A10 Murine anti- 64F1F8 2.0 Weakly positive Negative Negative 64F1 Murine anti- 7E5-2A9 2.9 Negative Negative Negative 7E5 Murine anti- 43H6A1 4.0 Negative Negative Negative 43H6 Murine anti- 46C4A6 13.3 Negative Negative Negative 46C4 Murine anti- 49G12G5 7.9 Negative Negative Negative 49G12 Murine anti- 51G7C2 26.2 Negative Negative Negative 51G7 Murine anti- 53D8H4 6.8 Negative Negative Negative 53D8 Murine anti- 54D2G10 19.5 Negative Negative Negative 54D2 Murine anti- 56F10G2 12.3 Negative Negative Negative 56F10 Murine anti- 59G3F12 40.9 Negative Negative Negative 59G3 Murine anti- 64B11F2 25.3 Negative Negative Negative 64B11
Example 4 Cell Killing Activity Experiment of EGFRvIII Antibody-Drug Conjugate
[0229] The antibody is dialyzed with a sodium borate buffer at Ph 6.5 to 8.5, and then a certain proportion of TCEP is added thereto (with TCEP/antibody ratio being 2-10); reduction is performed for 2 to 4 hours at room temperature or 37 C., and excess TCEPs are removed through a G25 desalting filler, and a certain proportion of MC-MMAF (with drug/antibody ratio being 5-20) is added and reacted for 4 hours. Then cysteine is added to neutralize excess drugs, and excess small molecules are removed through G25. A purified antibody-drug conjugate is afforded (for the coupling method, see Doronina, 2006, Bioconjugate Chem. 17, 114-124). The cytotoxic activity is analyzed after HIC analysis of drug cross-linking rate, purity and other parameters. For comparison, the drug cross-linking rate of all antibody conjugates is 8.
[0230] The resulting antibody conjugates are respectively subjected to gradient dilution with a complete medium, and a 96-well cell culture plate is added with 90 L of the U87MG-EGFRvIII cell suspension at 1000 cells/well, and to each well is further added 10 L of different concentrations of antibody-drug conjugate dilutions; after continuing the culture for 5 days, the cell viability is detected using the CellTiter-Glo kit (purchased from Promega, and the usage method refers to the product instructions).
[0231] The results are as shown in table 12 and
TABLE-US-00014 TABLE 12 Specific killing effect of antibody conjugates on EGFRvIII positive cells detected by a cell killing experiment IC50, nM Antibody U87MG- Antibody conjugate clone number DAR hEGFRvIII A431 HEB U87MG Murine anti- 75G7C6 8 0.27 Weakly Negative Negative 75G7-MMAF positive Murine anti- 63A10A7 8 0.01 Weakly Negative Negative 63A10-MMAF positive Murine anti- 64F1F8 8 0.01 Weakly Negative Negative 64F1-MMAF positive Murine anti- 7E5-2A9 8 0.02 Negative Negative Negative 7E5-MMAF Murine anti- 43H6A1 8 0.01 Negative Negative Negative 43H6-MMAF Murine anti- 46C4A6 8 0.15 Negative Negative Negative 46C4-MMAF Murine anti- 49G12G5 8 0.02 Negative Negative Negative 49G12-MMAF Murine anti- 53D8H4 8 0.04 Negative Negative Negative 53D8-MMAF Murine anti- 56F10G2 8 0.15 Negative Negative Negative 56F10-MMAF Murine anti- 59G3F12 8 0.06 Negative Negative Negative 59G3-MMAF Murine anti- 64B11F2 8 0.04 Negative Negative Negative 64B11-MMAF
Example 5 Epitope Analysis of EGFRvIII Antibody
A. Analysis of Epitope by a Competitive ELISA Method (Epitope Binning)
[0232] In order to identify the binding sites of antibodies to antigens, EGFRvIII antibodies are grouped using a competitive ELISA method.
[0233] The purified antibodies to be tested are diluted with PBS to 1 g/mL, coated onto a 96-well high-adsorption ELISA plate at 50 L/well at 4 C. overnight, and then blocked with 250 L of the blocking solution [PBS containing 0.01% (v/v) Tween20 and 1% (w/w) BSA] for one hour at room temperature, and each well is added with 0.05 g/mL of the biotin-labeled recombinant EGFRvIII protein. Simultaneously, 5 g/mL of the competitive antibody (i.e., the purified EGFRvIII antibody obtained in example 2) is added thereto, and incubated at 25 C.37 C. for 1 to 2 hours. The plate is washed 3 times with the plate washing solution [PBS containing 0.01% (v/v) Tween20], and added with HRP (horseradish peroxidase) labeled streptavidin (purchased from Sigma). The plate is incubated for 0.5 hour at 37 C., and then washed 3 times with the plate washing solution [PBS containing 0.01% (v/v) Tween20]. The plate is added with 100 L of TMB substrate/well, incubated for 30 minutes at room temperature, and then added with 100 L of stopping solution (1.0N HCl)/well. A450 nm value is read using an ELISA plate reader (SpectraMax 384 plus, purchased from Molecular Device), and the results are shown in
TABLE-US-00015 TABLE 13-1 Analysis of epitope by a competitive ELISA method (epitope binning) Control Control Competition antibody antibody Murine Murine Murine Murine rate 01 02 anti-7E5 anti-64Fl anti-63A10 anti-75G7 Control 66% 0% 4% 2% 5% 16% antibody 01 Control 2% 91% 81% 74% 37% 51% antibody 02 Murine anti- 2% 66% 85% 71% 70% 23% 7E5 Murine anti- 5% 69% 80% 72% 81% 13% 64F1 Murine anti- 3% 79% 82% 73% 89% 20% 63A10 Murine anti- 5% 2% 3% 3% 9% 67% 75G7
[0234] Competitive ELISA results show that epitopes of control antibody 02 and murine anti-7E5, -63A10 and -64F1 are competitive and should be the same or similar epitopes. Since the murine anti-75G7 is not competitive with control antibody 02, the epitope of the murine anti-75G7 should be different from that of the control antibody 02, wherein the control antibody 01 is AMG-595 (purchased from Amgen), and the control antibody 02 is ABT-414 (purchased from Abbvie).
B. Analysis of Epitope by a Polypeptide Dot Matrix ELISA Method (Epitope Mapping)
[0235] In order to determine the specific antibody binding epitopes of 4 candidate antibodies of the present invention, a polypeptide dot matrix ELISA method is used to analyze the antigen binding epitopes of the antibodies. A polypeptide of 16 amino acids in length is synthesized by using amino acids at positions 1 to 50 of the EGFRvIII protein as a template, starting from the N-terminus, and moving in a manner of overlapping 15 amino acids. The C-terminus of the polypeptide is added with a biotin label. The polypeptide is synthesized by GL Biochem, with 35 biotin labeled polypeptides synthesized in total. The detailed sequences are shown in table 13-2.
TABLE-US-00016 TABLE 13-2 Analysis of epitope by a polypeptide dot matrix ELISA method Chimeric Chimeric Chimeric Control Control antibody antibody antibody Polypeptides antibody 01 antibody 02 64F1 63A10 75G7 hIgG pep01 LEEKKGNYVV 3.62 3.59 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.08 0.07 TDHGSC-K (Biotin) pep02 EEKKGNYVVT 3.32 3.17 0.05 0.05 0.05 0.05 0.05 0.07 0.05 0.05 0.06 0.06 DHGSCV-K (Biotin) pep03 EKKGNYVVTD 0.14 0.10 0.05 0.05 0.05 0.05 0.32 0.05 0.12 0.06 0.17 0.10 HGSCVR-K (Biotin) pep04 KKGNYVVTD 0.12 0.06 0.06 0.05 0.05 0.56 0.06 0.06 0.06 0.05 0.05 0.06 HGSCVRA-K (Biotin) pep05 KGNYVVTDH 0.05 0.15 0.05 0.05 0.05 0.82 0.15 0.17 0.06 0.44 0.05 0.06 GSCVRAC-K (Biotin) pep06 GNYVVTDHGS 0.19 0.06 0.09 0.05 0.05 0.25 0.05 0.07 0.14 0.10 0.07 0.05 CVRACG-K (Biotin) pep07 NYVVTDHGSC 0.23 0.06 0.05 0.05 0.05 0.12 0.05 0.52 0.31 0.05 0.05 0.05 VRACGA-K (Biotin) pep08 YVVTDHGSCV 0.08 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 RACGAD-K (Biotin) pep09 VVTDHGSCVR 0.05 0.05 0.06 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.05 ACGADS-K (Biotin) pep10 VTDHGSCVRA 0.05 0.08 0.07 0.05 0.05 0.05 0.05 0.05 0.09 0.05 0.05 0.06 CGADSY-K (Biotin) pep11 TDHGSCVRAC 0.05 0.09 0.06 0.06 0.05 0.05 0.05 0.05 0.08 0.08 0.05 0.06 GADSYE-K (Biotin) pep12 DHGSCVRACG 0.09 0.06 0.06 0.06 0.05 0.05 0.05 0.06 0.07 0.11 0.05 0.05 ADSYEM-K (Biotin) pep13 HGSCVRACGA 0.06 0.08 0.08 0.05 0.05 0.07 0.06 0.07 0.07 0.10 0.06 0.06 DSYEME-K (Biotin) pep14 GSCVRACGAD 0.09 0.11 0.08 0.07 0.08 0.09 0.08 0.23 0.18 0.12 0.07 0.07 SYEMEE-K (Biotin) pep15 SCVRACGADS 0.06 0.05 0.05 0.06 0.09 0.11 0.14 0.11 0.06 0.11 0.05 0.06 YEMEED-K (Biotin) pep16 CVRACGADSY 0.06 0.05 0.10 0.05 0.05 0.08 0.05 0.05 0.05 0.05 0.05 0.05 EMEEDG-K (Biotin) pep17 VRACGADSYE 0.09 0.05 0.05 0.06 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 MEEDGV-K (Biotin) pep18 RACGADSYEM 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.05 0.05 0.05 0.05 0.05 EEDGVR-K (Biotin) pep19 ACGADSYEME 0.15 0.09 0.31 0.11 0.05 0.05 0.11 0.15 0.18 0.17 0.05 0.05 EDGVRK-K (Biotin) pep20 CGADSYEMEE 0.22 0.13 3.47 3.49 1.31 1.88 1.95 2.02 0.16 0.17 0.12 0.10 DGVRKC-K (Biotin) pep21 GADSYEMEED 0.15 0.31 0.32 0.43 0.05 0.06 0.21 0.26 0.19 0.18 0.06 0.06 GVRKCK-K (Biotin) pep22 ADSYEMEEDG 0.12 0.15 2.05 1.90 0.08 0.08 1.02 1.18 0.12 0.13 0.06 0.06 VRKCKK-K (Biotin) pep23 DSYEMEEDGV 0.06 0.06 0.19 0.16 0.07 0.07 0.19 0.20 0.05 0.05 0.06 0.06 RKCKKC-K (Biotin) pep24 SYEMEEDGVR 0.05 0.05 0.11 0.09 0.06 0.06 0.09 0.08 0.05 0.05 0.06 0.06 KCKKCE-K (Biotin) pep25 YEMEEDGVRK 0.06 0.06 0.07 0.07 0.06 0.06 0.06 0.06 0.06 0.05 0.06 0.06 CKKCEG-K (Biotin) pep26 EMEEDGVRKC 0.25 0.28 0.40 0.33 0.05 0.08 0.33 0.33 0.20 0.33 0.32 0.06 KKCEGP-K (Biotin) pep27 MEEDGVRKC 0.38 0.29 0.38 0.32 0.05 0.26 0.45 0.31 0.28 0.29 0.27 0.07 KKCEGPC-K (Biotin) pep28 EEDGVRKCKK 0.06 0.12 0.09 0.07 0.05 0.06 0.06 0.06 0.06 0.06 0.07 0.07 CEGPCR-K (Biotin) pep29 EDGVRKCKKC 0.06 0.08 0.07 0.07 0.06 0.06 0.06 0.05 0.05 0.09 0.07 0.07 EGPCRK-K (Biotin) pep30 DGVRKCKKCE 0.07 0.07 0.08 0.07 0.06 0.06 0.06 0.06 0.09 0.07 0.08 0.07 GPCRKV-K (Biotin) pep31 GVRKCKKCEG 0.06 0.07 0.07 0.07 0.06 0.06 0.06 0.06 0.06 0.06 0.07 0.08 PCRKVC-K (Biotin) pep32 VRKCKKCEGP 0.06 0.07 0.07 0.07 0.06 0.06 0.13 0.06 0.06 0.06 0.07 0.08 CRKVCN-K (Biotin) pep33 RKCKKCEGPC 0.08 0.10 0.08 0.07 0.07 0.08 0.07 0.07 0.07 0.07 0.07 0.06 RKVCNG-K (Biotin) pep34 KCKKCEGPCR 0.07 0.09 0.07 0.09 0.07 0.07 0.07 0.07 0.06 0.06 0.07 0.08 KVCNGI-K (Biotin) pep35 CKKCEGPCRK 0.07 0.07 0.07 0.07 0.09 0.07 0.07 0.07 0.07 0.07 0.07 0.07 VCNGIG-K (Biotin)
[0236] The results of the polypeptide dot matrix ELISA show that the control antibody 01 and murine anti-7E5, murine anti-64F1 and murine anti-63A10 have the same or similar epitope, i.e., CGADSYEMEEDGVRKC. Since the murine anti-75G7 does not bind to the 35 polypeptides, the epitope of the murine anti-75G7 should be different from that of the control antibody 01 and that of the control antibody 02, wherein the control antibody 01 is AMG-595 (purchased from Amgen), and the control antibody 02 is ABT-414 (purchased from Abbvie). Therefore, the epitope of the murine anti-75G7 needs to be further verified by experiments.
Example 6 Analysis of Binding Specificity of EGFRvIII Antibody
[0237] The EGFRvIII antibody can specifically recognize the activated EGFR, i.e., the mutant EGFRvIII and overexpressed wild-type EGFR in tumor tissues, but does not recognize the wild-type EGFR in normal tissues. Therefore, the antibody has an excellent target specificity, which can effectively avoid the harm of the antibody and conjugates thereof to human normal tissues, and effectively gather the antibody and conjugates thereof at the location of the tumor, thereby accurately and effectively killing tumor tissues, and significantly reducing the toxic and side effects of drugs on other normal organs.
A. Binding Specificity of EGFRvIII Antibody to Human Normal Tissue Cell Lines Detected by FACS
[0238] Seven human normal tissue cell lines are respectively selected from different tissues, and see table 14 for details. The binding of the EGFRvIII antibody to the seven human normal tissue cell lines is detected and analyzed by FACS. The results as shown in
TABLE-US-00017 TABLE 14 Cell lines derived from 7 different human normal tissues Cell line name Tissue source HFF-1 Skin HFL-I Lung QSG-7701 Liver HEEC Esophagus HEB Brain WPMY-1 Prostate MCF-10A Breast
B. Binding Specificity of the EGFRvIII Antibody Detected by Immunohistochemical Staining (IHC)
[0239] The human glioma tissue chip (Cat #GL805d) and human multi-organ normal tissue chip (Cat #FDA999n) are both purchased from Alenabio, Co., Ltd. (Xi'an).
[0240] The experimental procedure of the immunohistochemical staining is as follows:
[0241] 1) Baking the slices at 60 C. for 2 hours. The purpose of baking the slices is to firmly stick the tissue slices with paraffin on the slide to prevent the slices from falling off during the staining. The purpose is achieved by placing the slices in an incubator at 56 C. to 60 C. for at least 1 hour; however, the usual temperature of baking slices is 8 C. to 60 C. and the time thereof is 2 to 6 hours. Since high-temperature drying may accelerate the oxidation of antigens in the tissues, baking slices at a high temperature has a destructive effect on antigens.
[0242] 2) Performing deparaffinage and hydration. Soaking is carried out in xylene I and II for 15 to 30 minutes to remove the paraffin from the tissues. However, the xylene entering human tissues is immiscible with a water-soluble staining solution, which makes it necessary to gradually replace the xylene in the tissues with a descending gradient of ethanol. The slices are soaked respectively in 100% ethanol I and II for 5 minutes and respectively in 95%, 90%, 80% and 70% ethanol for 2 minutes, rinsed with PBS 3 times, with 3 minutes each time, and placed in distilled water for later use.
[0243] 3) Antigen repair. The tissue chip is placed in an EDTA solution (pH 9.0) and repair is performed at high temperature for 5 minutes.
[0244] 4) Blocking endogenous peroxidase. The tissue chip is placed in 3% H.sub.2O.sub.2 at room temperature for 5 minutes.
[0245] 5) Blocking non-immune serum. The tissue chip is placed in 5% FBS at room temperature for 15 minutes. Antibodies can be adsorbed by charged collagen and connective tissue components in tissue slices, which causes background coloring, and in order to prevent this situation, it is best to select the non-immune serum derived from the same species as the secondary antibody to block the charge before treating the slices with specific antibodies, which prevents the primary antibody from binding to the charge, and inhibits the non-specific background coloring. A common method is to use 2% to 10% sheep serum or 2% to 5% bovine serum albumin for reaction at room temperature for 10 to 30 minutes.
[0246] 6) Incubating the primary antibody and placing the primary antibody at room temperature for 1 hour.
[0247] 7) Incubating the secondary antibody and placing the secondary antibody at room temperature for 0.5 hour.
[0248] 8) Allowing DAB to develop color and placing same at room temperature for 5 minutes.
[0249] 9) Counterstaining nucleus with hematoxylin and placing same at room temperature for 10 seconds.
[0250] 10) Sealing for detection.
[0251] The results of the immunohistochemical staining are shown in
TABLE-US-00018 TABLE 15 Response rate of EGFR antibody and EGFRvIII antibody to human glioma tissue chips Anti-EGFR anti-EGFRvIII anti-EGFRvIII anti-EGFRvIII anti-EGFRvIII 70 GBM (murine (murine anti- (murine anti- (murine anti- (murine anti- samples anti-80E11) 63A10) 64F1) 7E5-2) 75G7) Positive 70 24 24 24 / samples % positive 100% 34% 34% 34% /
[0252] It is proved in the FACS and IHC experiments that the EGFRvIII antibody can specifically recognize the EGFR in tumor tissues, but does not recognize the EGFR in normal tissues. Therefore, the antibody has an excellent target specificity, which can effectively avoid the harm of the antibody and conjugates thereof to human normal tissues, and effectively gather the antibody and conjugates thereof at the location of the tumor, thereby accurately and effectively killing tumor tissues, and significantly reducing the toxic and side effects of drugs on other normal organs.
Example 7 Determination of Amino Acid Sequences of Light and Heavy Chain Variable Regions
[0253] Total RNA isolation: 510.sup.7 hybridoma cells obtained in example 1 are collected by centrifugation, added with 1 mL Trizol and mixed evenly, transferred to a 1.5 mL centrifuge tube, and placed at room temperature for 5 minutes. The tube is added with 0.2 mL of chloroform, shaken for 15 seconds, standing for 10 minutes, and then centrifuged at 4 C. and at 12,000 g for 5 minutes, and the supernatant is taken and transferred to a new 1.5 mL centrifuge tube. 0.5 mL isopropanol is added, the liquid in the tube is mixed homogeneously and gently, then the tube is standing at room temperature for 10 minutes, is centrifuged at 4 C. and at 12000 g for 15 minutes, then the supernatant is discarded. After adding 1 mL of 75% (v/v) ethanol, the precipitate is gently washed; then the tube is centrifuged at 4 C. and at 12000 g for 5 minutes to discard the supernatant; the precipitate is dried in the air, and then the DEPC-treated H.sub.2O is added to perform dissolution (promoting the dissolution at 55 C. in water bath for 10 minutes) so as to obtain total RNA.
[0254] Reverse transcription and PCR: 1 g of total RNA is taken to formulate a 20 L system, to which a reverse transcriptase is added, for reacting at 42 C. for 60 minutes and at 7 C. for 10 minutes so as to stop the reaction. A 50 L PCR system is formulated, comprising 1 L cDNA, 25 pmol of each primer, 1 L DNA polymerase, a corresponding buffer system, and 250 mol dNTPs; the PCR program is set: pre-denaturating at 95 C. for 3 minutes; denaturating at 95 C. for 30 seconds; annealing at 55 C. for 30 seconds; extending at 72 C. for 35 seconds; after 35 cycles, further extending at 72 C. for 5 minutes to obtain a PCR product. The kit used for reverse transcription is PrimeScript RT Master Mix, purchased from Takara (Cat #RR036); and the kit used for PCR includes a Q5 ultra-fidelity enzyme, purchased from NEB (Cat #M0492).
[0255] Cloning and sequencing: 5 L of the PCR product is taken for an agarose gel electrophoresis detection, and the column recovery kit is used to purify the positive test samples, wherein the recovery kit is NucleoSpin Gel & PCR Clean-up, purchased from MACHEREY-NAGEL (Cat #740609). The ligation reaction is performed as follows: 50 ng of the sample, 50 ng of the T vector, 0.5 L of the ligase, 1 L of the buffer, and 10 L of the reaction system are reacted at 16 C. for half an hour to obtain a ligation product. The ligated kit is a T4 DNA ligase, purchased from NEB (Cat #M0402); 5 L of the ligation product is added to 100 L of competent cells (Ecos 101 competent cells, purchased from Yeasten, Cat #FYE607), put into an ice bath for 5 minutes, then heat shocked in 42 C. water bath for 1 minute, put back on ice for 1 minute, then added with 650 L of the antibiotic-free SOC medium, and recovered at a speed of 200 RPM on a shaker at 37 C. for 30 minutes. 200 L of the mixture is taken and coated on an LB solid medium containing antibiotics, and incubated in a 37 C. incubator overnight. The next day, primers M13F and M13R on the T vector are used to formulate 30 L of the PCR system for a colony PCR, wherein the colony is dipped with a pipette tip to the PCR reaction system, and then is blown and sucked; and 0.5 L is sucked out and placed on another LB solid culture dish containing 100 nM ampicillin to preserve the strain. After the PCR reaction is completed, 5 L is taken for an agarose gel electrophoresis detection, and the positive samples are sequenced and analyzed (wherein the CDR is divided according to the Chothia definition system). The sequencing results are shown in tables 16 and 17.
TABLE-US-00019 TABLE 16 Numbers of amino acid sequences of EGFRvIII antibody Heavy chain protein Light chain protein Clone Variable Variable number region CDR1 CDR2 CDR3 region CDR1 CDR2 CDR3 75G7C6 1 2 3 4 5 6 7 8 63A10A7 9 10 11 12 13 14 15 16 64F1F8 17 18 19 20 21 22 23 24 7E5-2A9 25 26 27 28 29 30 31 32 43H6A1 33 34 35 36 37 38 39 40 46C4A6 41 42 43 44 45 46 47 48 49G12G5 49 50 51 52 53 54 55 56 51G7C2 57 58 59 60 61 62 63 64 53D8H4 65 66 67 68 69 70 71 72 54D2G10 73 74 75 76 77 78 79 80 56F10G2 81 82 83 84 85 86 87 88 59G3F12 89 90 91 92 93 94 95 96 64B11F2 97 98 99 100 101 102 103 104
[0256] The numbers in table 17 are the sequence numbers in the sequence listing. For example, the amino acid sequence of the heavy chain protein variable region of 43H6A1 is SEQ ID No. 1, and the amino acid sequence of the CDR1 in the heavy chain protein variable region of 43H6A1 is SEQ ID No. 2.
TABLE-US-00020 TABLE 17 Numbers of nucleotide sequences of EGFRvIII antibody Clone Heavy chain protein Light chain protein number variable region variable region 75G7C6 105 106 63A10A7 107 108 64F1F8 109 110 7E5-2A9 111 112 43H6A1 113 114 46C4A6 115 116 49G12G5 117 118 51G7C2 119 120 53D8H4 121 122 54D2G10 123 124 56F10G2 125 126 59G3F12 127 128 64B11F2 129 130
[0257] The numbers in table 18 are the sequence numbers in the sequence listing. For example, the nucleotide sequence encoding the heavy chain protein variable region of 75G7C6 is SEQ ID No. 105.
Example 8 Production and Purification of EGFRvIII Human-Murine Chimeric Antibody
[0258] 1) Plasmid construction and preparation: The heavy chain variable region sequence of the above-mentioned murine lead antibody is recombined into an expression vector containing a signal peptide and a human heavy chain antibody IgG1 constant region (wherein the expression vector is purchased from Invitrogen, and the recombination steps are also completed by Shanghai ChemPartner Co., Ltd.), and the light chain variable region sequence of the EGFRvIII antibody is recombined into an expression vector containing a signal peptide and a human antibody light chain kappa constant region (wherein the expression vector is purchased from Invitrogen, and the recombination steps are also completed by Shanghai ChemPartner Co., Ltd), thereby obtaining recombinant plasmids (for the experimental principles and steps of the above-mentioned plasmid recombination, see Molecular Cloning: A Laboratory Manual (Third Edition), (US) J. Sambrook et al.) which are verified by sequencing. An alkaline lysis kit (purchased from MACHEREY-NAGEL) is used for a medium-amount extraction of recombinant plasmids having a high purity, wherein the recombinant plasmids have a mass of at least 500 g and filtered through a 0.22 m filter membrane (purchased from Millopore) for transfection.
[0259] 2) Cell transfection: 293E cells (purchased from Invitrogen) are cultured in a Freestyle 293 expression medium (purchased from Invitrogen). The shaker is set to 37 C., 130 RPM and 8% CO.sub.2 (v/v). During transfection, the Freestyle 293 expression medium is added with 10% (v/v) F68 (purchased from Invitrogen) to a final F68 concentration of 0.1% (v/v), thereby obtaining a Freestyle 293 expression medium containing 0.1% (v/v) F68 (i.e., a medium A). 5 mL of the medium A and 200 g/mL PEI (purchased from Sigma) are mixed evenly to obtain a medium B. 5 mL of the medium A and 100 g of the recombinant plasmids (herein referred to the mixed recombinant plasmids obtained by mixing the above-mentioned heavy chain recombinant plasmid and light chain recombinant plasmid in a conventional equal proportion) obtained in step 1) are mixed evenly to obtain a medium C. After 5 minutes, the medium B and the medium C are combined and mixed evenly, and allowed to stand for 15 minutes to obtain a mixed solution D. 10 mL of the mixed solution D is slowly added to 100 mL of the Freestyle 293 expression medium containing 293E cells until the cell density of 293E is 1.510.sup.6 cells/mL, wherein the addition is performed with shaking to avoid excessive concentration of PEI, and the mixture is placed in a shaker for culture. The next day peptone is added to a final concentration of 0.5% (w/v). On days 5 to 7, the antibody titer of the culture broth is measured. On days 6 and 7, the supernatant is collected by centrifugation (3500 RPM, 30 minutes) and filtered through a 0.22 m filter membrane to obtain the filtered cell supernatant for purification.
[0260] 3) Antibody purification: The continuously produced endotoxin-free chromatography columns and protein A fillers are treated with 0.1 M NaOH for 30 minutes or rinsed with 5 column volumes of 0.5 M NaOH; the long-term unused column materials and chromatography columns are soaked with 1 M NaOH for at least 1 hour, rinsed with endotoxin-free water to neutrality, and washed with 10-fold column volumes of 1% Triton X100. 5 column volumes of PBS are used for equilibration, and the filtered cell supernatant is loaded onto the column, and the flow-through fluid is collected if necessary. After loaded onto the column, 5-fold column volumes of PBS are used for washing. 5-fold column volumes of 0.1 M Glycine-HCl (pH 3.0) is used for eluting, and the eluate is collected and neutralized with 1/10 volume of 1 M Tris-HCl (1.5 M NaCl) (pH 8.5). The antibodies are harvested, and then dialyzed in 1PBS overnight to avoid endotoxin contamination. After dialysis, the concentration is measured using a spectrophotometer or a kit; the purity of the antibodies is measured using HPLC-SEC; and the endotoxin content of the antibodies is detected using an endotoxin detection kit (purchased from Lonza).
[0261] Purified EGFRvIII chimeric antibodies 75G7, 63A10, 64F1 and 7E5 are obtained respectively.
[0262] The purified EGFRvIII chimeric antibody is detected and analyzed for protein concentration (A280/1.4), purity, endotoxin (Lonza kit), etc. The results as shown in table 18 indicate that the endotoxin concentration of the final antibody product is within 1.0 EU/mg.
TABLE-US-00021 TABLE 18 Quality control of purified EGFRvIII chimeric antibody Protein Clone Antibody concentration Endotoxin Antibody name number purity (mg/ml) (EU/mg) Chimeric antibody 75G7C6 >95% 1.44 <1.0 75G7 Chimeric antibody 63A10A7 >95% 1.26 <1.0 63A10 Chimeric antibody 64F1F8 >95% 1.02 <1.0 64F1 Chimeric antibody 7E5-2A9 >95% 1.81 <1.0 7E5
Example 9 Detection and Identification of EGFRvIII Chimeric Antibody
A. Binding of the Antibody to an EGFRvIII Protein Detected by an Enzyme-Linked Immunosorbent Assay (ELISA)
[0263] The purified EGFRvIII chimeric antibody obtained in example 2 is reacted with a human EGFRvIII-hFc protein.
[0264] The purified immunogen A (EGFRvIII-hFc) obtained in example 1 (for the preparation method, see step (I) in example 1) is diluted with PBS to a final concentration of 1.0 g/mL, and then added to a 96-well ELISA plate at 100 l/well. The plate is sealed with a plastic film and incubated at 4 C. overnight. The next day, the plate is washed twice with a plate washing solution [PBS+0.01% (v/v) Tween20], and added with a blocking solution [PBS+0.01% (v/v) Tween20+1% (w/w) BSA] for blocking 2 hours at room temperature. The blocking solution is discarded, and the purified EGFRvIII chimeric antibody obtained in example 2 is added at 100 l/well. The plate is incubated for 2 hours at 37 C., and then washed 3 times with the plate washing solution [PBS+0.01% (v/v) Tween20]. The plate is added with an HRP (horseradish peroxidase) labeled secondary antibody (purchased from Sigma), incubated at 37 C. for 2 hours, and then washed 3 times with the plate washing solution [PBS+0.01% (v/v) Tween20]. The plate is added with 100 l of TMB substrate/well, incubated for 30 minutes at room temperature, and then added with 100 l of stopping solution (1.0N HCl)/well. A450 nm value is read using an ELISA plate reader (SpectraMax 384plus, purchased from Molecular Device), and the results are shown in
TABLE-US-00022 TABLE 19 Binding reaction of EGFRvIII chimeric antibody with human EGFRvIII-hFc protein detected by ELISA OD450 nm Clone Antibody concentration (nM) Antibody name number 100 10 1 0.1 0.01 0.001 0.0001 Blank Chimeric antibody 75G7C6 3.44 3.41 2.69 0.65 0.15 0.08 0.07 0.08 75G7 Chimeric antibody 63A10A7 3.55 3.55 3.33 1.05 0.37 0.11 0.07 0.06 63A10 Chimeric antibody 64F1F8 3.70 3.78 3.72 2.23 0.35 0.11 0.08 0.08 64F1 Chimeric antibody 7E5-2A9 3.60 3.63 3.45 1.18 0.19 0.08 0.09 0.07 7E5 Control antibody Positive 3.44 3.41 2.69 0.65 0.15 0.08 0.07 0.08 control hIgG IgG 0.11 0.07 0.06 0.1 0.06 0.06 0.07 0.06 control
B. Binding of Antibody to EGFRvIII Expressing Cell Detected by Fluorescence Activated Cell Sorting (FACS)
[0265] The desired cells are subjected to an expanded culture to 90% confluence in a T-75 cell culture flask; the medium is sucked out, and the cells are washed twice with a HBSS buffer (Hanks Balanced Salt Solution) (purchased from Invitrogen), and then treated with an enzyme-free cell dissociation solution (Versene solution: purchased from Life Technology) and collected. The cells are washed twice with the HBSS buffer and are counted, and then the cells are diluted with the HBSS buffer to 210.sup.6 cells/ml, added with 1% goat serum blocking solution, with the percentage being the mass percentage, and incubated on ice for 30 minutes, and then centrifugally washed twice with the HBSS buffer. The collected cells are suspended to 210.sup.6 cells/mL with an FACS buffer (HBSS+1% BSA, with the percentage being the mass percentage), added to a 96-well FACS reaction plate at 100 L/well, then added with 100 L/well of the purified EGFRvIII antibody (samples to be tested) obtained in example 2, and incubated on ice for 2 hours. The plate is centrifugally washed twice with the FACS buffer, added with 100 L/well of a fluorescence (Alexa 488) labeled secondary antibody (purchased from Invitrogen), and then incubated on ice for 1 hour. The plate is centrifugally washed 3 times with the FACS buffer, added with 100 L/well of a fixing solution [4% (v/v) paraformaldehyde] for suspending the cells, and centrifugally washed twice with the FACS buffer after 10 minutes. The cells are suspended in 100 L of the FACS buffer, and the results are detected and analyzed by FACS (FACS Calibur, purchased from BD). Data analysis is performed by using the software (CellQuest) to obtain the mean fluorescence intensity (MFI) of the cells. Data is further analyzed by using the software (GraphPad Prism5) to perform data fitting and calculate EC50. The analysis results as shown in table 20 and
TABLE-US-00023 TABLE 20 Binding reaction of EGFRvIII chimeric antibody with U87MG- hEGFRvIII, A431, U87MG and HEB cells detected by FACS Antibody EC50, nM Antibody clone U87MG- name number hEGFRvIII A431 HEB U87MG Chimeric antibody 75G7C6 6.1 13.4 Negative Negative 75G7 Chimeric antibody 63A10A7 2.2 696.0 Negative Negative 63A10 Chimeric antibody 64F1F8 2.1 180.4 Negative Negative 64F1 Chimeric antibody 7E5-2A9 1.9 Negative Negative Negative 7E5
Example 10 Cell Killing Activity Experiment of EGFRvIII Chimeric Antibody-Drug Conjugate
[0266] The antibody is dialyzed with a sodium borate buffer at Ph 6.5 to 8.5, and then a certain proportion of TCEP is added thereto (with TCEP/antibody ratio being 2-10); reduction is performed for 2 to 4 hours at room temperature or 37 C., and excess TCEPs are removed through a G25 desalting filler, and a certain proportion of MC-MMAF (with drug/antibody molar ratio being 4.5) is added and reacted for 4 hours. Then cysteine is added to neutralize excess drugs, and excess small molecules are removed through G25. A purified antibody-drug conjugate is afforded (for the coupling method, see Doronina, 2006, Bioconjugate Chem. 17, 114-124). The cytotoxic activity is analyzed after HIC analysis of drug cross-linking rate, purity and other parameters. For comparison, the drug cross-linking rate of all antibody conjugates is 8 [the DAR (drug-antibody coupling ratio) is adjusted through experiments to reach the preset value, and the exact DAR is detected and determined later].
[0267] The resulting antibody conjugates are respectively subjected to gradient dilution with a complete medium, and a 96-well cell culture plate is added with 90 L of the U87MG-EGFRvIII cell suspension at 1000 cells/well, and to each well is further added 10 L of different concentrations of antibody-drug conjugate dilutions; after continuing the culture for 5 days, the cell viability is detected using the CellTiter-Glo kit (purchased from Promega, and the usage method refers to the product instructions).
[0268] The results are as shown in table 21 and
TABLE-US-00024 TABLE 21 Killing effect of chimeric antibody conjugates on EGFRvIII positive cells detected by a cell killing experiment IC50, nM Antibody U87MG- Antibody conjugate clone number DAR hEGFRvIII A431 HEB U87MG Chimeric antibody 75G7C6 3.40 0.27 Weakly Negative Negative 75G7-MMAF positive Chimeric antibody 63A10A7 3.10 0.01 Weakly Negative Negative 63A10-MMAF positive Chimeric antibody 64F1F8 4.05 0.01 Weakly Negative Negative 64F1-MMAF positive Chimeric antibody 7E5-2A9 3.51 0.04 Negative Negative Negative 7E5-MMAF
Example 11 In Vivo Pharmacodynamic Study of EGFRvIII Chimeric Antibody-Drug Conjugate
[0269] Since the expression of EGFRvIII genes may gradually be lost during the in vitro cultivation of tumor cells, there is no endogenous tumor cell line that stably expresses the EGFRvIII gene. Therefore, the U87MG-EGFRvIII recombinant cell line is constructed and the U87MG-EGFRvIII subcutaneous xenograft tumor model is established to test the in vivo efficacy of the chimeric antibody-drug conjugate.
[0270] See example 1 for the construction method of the U87MG-EGFRvIII recombinant cell line. 200 l (110.sup.6 cells) of the U87MG-EGFRvIII cell suspension is inoculated on the right back of Balb/c nude mice; after 7 days, tumor grew to 150-250 mm.sup.3, and then tumor-bearing mice with appropriate tumor volume are selected, randomly grouped according to the tumor volume before administering drugs per the established schedule. See table 22 for specific research plan and administration dose.
[0271] The experimental indicators are to investigate whether tumor growth may be inhibited, delayed or cured. The next day the diameter of the tumor is measured with a vernier caliper. The computational formula of the tumor volume is: V=0.5ab.sup.2, where a and b represent the long diameter and short diameter of the tumor, respectively.
[0272] The tumor suppressive effect (TGI) of the compounds is evaluated by TGI (%). TGI %=1(TRTVnTRTV1)/(CRTVnCRTV1)100% (TRTVn: RTV on the last day of the treatment group; CRTV: RTV in the negative control group). The computational formula of the relative tumor volume RTV is RTV=Vt/V0, wherein V0 is the tumor volume measured during administration in different cages (Day 0), and Vt is the tumor volume at each measurement. The percentage value of T/C (%) reflects the tumor growth inhibition rate; according to guiding principles regarding anti-tumor drugs of Center for Drug Evaluation, TGI %58% indicates that the drug is effective.
TABLE-US-00025 TABLE 22 In vivo pharmacodynamic study of EGFRvIII chimeric antibody-drug conjugates Numbers Dose of (unit: Injection Route of Administration Groups animals Treatment group mg/kg) volume administration schedule 1 8 Solvent control 10 l/g Tail vein Dosing every injection i.v 4 days x 4 times 2 8 Control antibody 3 10 l/g Tail vein Dosing every 02-MMAE injection i.v 4 days x 4 times 3 8 Control antibody 1 10 l/g Tail vein Dosing every 02-MMAE injection i.v 4 days x 4 times 4 8 Chimeric antibody 3 10 l/g Tail vein Dosing every 63A10-MMAE injection i.v 4 days x 4 times 5 8 Chimeric antibody 1 10 l/g Tail vein Dosing every 63A10-MMAE injection i.v 4 days x 4 times 6 8 Chimeric antibody 3 10 l/g Tail vein Dosing every 75G7-MMAE injection i.v 4 days x 4 times 7 8 Chimeric antibody 1 10 l/g Tail vein Dosing every 75G7-MMAE injection i.v 4 days x 4 times 8 8 Chimeric antibody 3 10 l/g Tail vein Dosing every 64F1-MMAE injection i.v 4 days x 4 times 9 8 Chimeric antibody 1 10 l/g Tail vein Dosing every 64F1-MMAE injection i.v 4 days x 4 times 10 8 Chimeric antibody 3 10 l/g Tail vein Dosing every 7E5-MMAE injection i.v 4 days x 4 times 11 8 Chimeric antibody 1 10 l/g Tail vein Dosing every 7E5-MMAE injection i.v 4 days x 4 times
[0273] The results are shown in
Example 12 Inhibition of Introduction of Mutations for Deamidation, Isomerization, and Hydrolysis Reactions
[0274] By analyzing the antibody sequences of 75G7C6 and 63A10A7, it is found that there is a possibility of deamidation reaction of NG at positions 54 and 55 of the CDR2 (SEQ ID No. 3) and isomerization of DG at positions 98 and 99 of the CDR3 (SEQ ID No. 4) in the heavy chain variable region (SEQ ID No. 1) of the 75G7C6 antibody. In order to suppress deamidation, isomerization and hydrolysis, upon calculation and analysis, the NG located in the CDR2 of the 75G7C6 antibody is mutated to NA, and the amino acid sequence of the heavy chain variable region of the 75G7C6 antibody after mutation is as shown in SEQ ID No. 179; or the NG is mutated to QG, and the amino acid sequence of the heavy chain variable region of the 75G7C6 antibody after mutation is as shown in SEQ ID No. 180. Moreover, the DG located in the CDR3 is mutated to SG, and the amino acid sequence of the heavy chain variable region of the 75G7C6 antibody after mutation is as shown in SEQ ID No. 181 (the corresponding nucleic acid sequence is as shown in SEQ ID No. 188); or the DG is mutated to EG, and the amino acid sequence of the heavy chain variable region of the 75G7C6 antibody after mutation is as shown in SEQ ID No. 182 (the corresponding nucleic acid sequence is as shown in SEQ ID No. 131); or the DG is mutated to DA, and the amino acid sequence of the heavy chain variable region of the 75G7C6 antibody after mutation is as shown in SEQ ID No. 183 (the corresponding nucleic acid sequence is as shown in SEQ ID No. 132). Amino acid modifications of removing asparagine residues and aspartoyl residues that are used as sites undergoing deamidation, are intended to be performed by means of the above-mentioned site-directed mutations.
[0275] After the site-directed mutant sequences of the heavy chain variable region of the chimeric 75G7C6 antibody are genetically synthesized, plasmid construction, cell transfection, and antibody purification are performed as described in example 8. The purified antibodies are identified for the mutant chimeric antibody binding activity by ELISA and FACS as described in example 9. ELISA identification results are shown in
TABLE-US-00026 TABLE 23 Binding reaction of c75G7C6 chimeric antibody mutant with human EGFRvIII-hFc protein detected by ELISA Maximum Antibody name Point mutation EC50 (unit: nM) OD450 value hIgG None None 0.08 c75G7C6 wt (NG & DG) 0.02 3.03 c75G7C6-1 NG/NA 0.02 3.44 c75G7C6-2 NG/QG 0.03 3.23 c75G7C6-3 DG/EG 0.54 1.86 c75G7C6-4 DG/SG 7.94 0.79 c75G7C6-5 DG/DA None 0.14
[0276] FACS identification results are as shown in
TABLE-US-00027 TABLE 24 Binding reaction of c75G7C6 chimeric antibody mutants with CHOk1-EGFRvIII cells detected by FACS Maximum mean fluorescence Antibody name Point mutation EC50 (unit: nM) intensity c75G7C6 Wild type 10.5 111769 (NG & DG) c75G7C6-1 NG/NA 7.452 109947 c75G7C6-2 NG/QG 11.64 111041 c75G7C6-3 DG/EG 24.82 94071 c75G7C6-4 DG/SG 24 52584 c75G7C6-5 DG/DA None 296 Negative None None 132.6 control hIgG
TABLE-US-00028 TABLE 25 Binding reaction of c75G7C6 chimeric antibody mutants with CHOk1-EGFR cells detected by FACS Maximum mean fluorescence Antibody name Point mutation EC50 (unit: nM) intensity c75G7C6 Wild type 14.22 137153 (NG & DG) c75G7C6-1 NG/NA 11.27 134462 c75G7C6-2 NG/QG 16.92 130112 c75G7C6-3 DG/EG 44.82 84723 c75G7C6-4 DG/SG None 8968 c75G7C6-5 DG/DA None 121 Negative None None 144 control hIgG
[0277] As can be seen from table 24 and
[0278] As can be seen from table 25 and
[0279] As can be seen from the above-mentioned ELISA and FACS results, the NG located at the CDR2 in the heavy chain variable region of the c75G7C6 can be mutated to NA or QG, and the binding activity in antigens of the mutated antibodies is not affected; however, whether the DG located in CDR3 is mutated to EG, SG or DA, it affects the binding of the antibodies to antigens, in which case mutation is not suitable. Therefore, the antibody c75G7C6-1 with NG of the CDR2 mutated to NA will be selected for follow-up study.
Example 13 Preparation of Humanized EGFRvIII Antibody
[0280] The heavy and light chain variable region templates of human germline antibodies that best match the non-CDR regions of the above-mentioned chimeric antibodies 63A10A7 and 75G7C6 are selected from the Germline database. The human acceptable sequences of the humanized EGFRvIII antibody are selected from human germline exons V.sub.H, J.sub.H, V.sub.k and J.sub.k sequences. The template of the heavy chain variable region of the 63A10A7 antibody is IGHV1-46*01 of the human germline antibody heavy chain exon V.sub.H, and J.sub.H-4 of the exon J.sub.H; the template of the light chain variable region is IGKV1-39*01 of the human germline antibody light chain exon V.sub.K, and J.sub.K-2 of the exon J.sub.K. The template of the heavy chain variable region of the 75G7C6 antibody is IGHV1-46*01 of the human germline antibody heavy chain exon V.sub.H, and J.sub.H-6b of the exon J.sub.H; and the template of the light chain variable region is IGKV3-11*01 of the human germline antibody light chain exon V.sub.K, and J.sub.K-4 of the exon J.sub.K.
[0281] The heavy chain and light chain CDRs of the chimeric antibodies 63A10A7 and 75G7C6 determined according to the Kabat definition are grafted into the selected human germline templates, respectively, to replace the CDR regions of the human germline templates so as to obtain a humanized antibody. Then, based on the three-dimensional structure of the murine antibody, the residues that embed residues and directly interact with the CDR regions, and the residues in framework regions that have an important influence on the conformations of VL and VH are subjected to back mutation to obtain a humanized antibody. Briefly, synthetic overlapping oligonucleotides across the humanized V.sub.H or V.sub.L domain are generated, and the PCR overlap extension is used to assemble each domain. The restriction site incorporated into the PCR product is used to directionally clone the V.sub.H domain into an expression vector containing a signal peptide and a human antibody heavy chain IgG1 constant region, and to directionally clone the VL domain into an expression vector containing a signal peptide and a human antibody light chain kappa constant region, thereby obtaining recombinant plasmids which are verified by sequencing; an alkaline lysis kit (purchased from MACHEREY-NAGEL) is used for a medium-amount extraction of the recombinant plasmids having a high purity, wherein the recombinant plasmids have a mass of at least 500 g and filtered through a 0.22 m filter membrane (purchased from Millopore) for transfection.
[0282] The sequence alignments of the heavy and light chain variable regions of the humanized anti-EGFRVIII antibody variants with the human germline heavy and light chain variable regions and the heavy and light chain variable regions of chimeric antibodies are as shown in
TABLE-US-00029 Humangermlineheavychainvariableregion templateIGHV1-46*01/JH4 IGHV1-46*01: (SEQIDNO.175) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGI INPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR: JH4: (SEQIDNO.189) YFDYWGQGTLVTVSS. Humangermlineheavychainvariableregion templateIGHV1-46*01/JH6b IGHV1-46*01: (SEQIDNO176) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGI INPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR JH6b: (SEQIDNO.190) YYYYYGMDVWGQGTTVTVSS Humangermlinelightchainvariableregion templateIGKV3-11*01/JK4 IGKV3-11*01: (SEQIDNO.177) EIVLTQSPATLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP: JK4: (SEQIDNO.191) LTFGGGTKVEIK. Humangermlinelightchainvariableregion templateIGKV1-39*01/JK2 IGKV1-39*01: (SEQIDNO.178) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP: JK2: (SEQIDNO.192) YTFGQGTKLEIK.
[0283] Several framework positions are selected to reintroduce mouse donor residues. Several humanized 75G7C6 and 63A10A7 variants may be generated by incorporating different combinations of mouse framework donor residues into the VH domain or of human CDR residues into the VL domain. Table 26 and table 27 summarize these variants. Table 26 and table 27 show the variable regions but not constant regions of these variants. In tables 26 and 24, beginning with c represents a chimeric antibody, and beginning with h represents a humanized antibody; in the column of donor framework residues and back mutations, for example, Q1E in the heavy chain variable region h75G7C6.VH of the humanized anti-EGFRvIII antibody 75G7C6 and variants thereof means that the amino acid at position 1 as shown in
TABLE-US-00030 TABLE 26 Human Human germline germline antibody Heavy antibody Light heavy chain Donor chain light chain Donor chain variable framework variable variable framework variable Heavy region residues region Light region residues region Antibody chain template and back SEQ ID chain template and back SEQ ID number name VH mutations NO. name Vk mutations NO. c75G7C6 VH None CDR 1 VL None CDR 5 h75G7C6-1 VH- IGHV1- CDR- 133 VL- IGKHV3- CDR- 137 g3 46*01/ grafted, Q1E, g0 11*01/ grafted, JH6b R38K, M48I, JK4 M70L, R72V, T74K h75G7C6-2 VH- IGHV1- CDR- 133 VL- IGKHV3- CDR- 138 g3 46*01/ grafted, Q1E, g1 11*01/ grafted, I2N, JH6b R38K, M48I, JK4 Y50F, F72Y M70L, R72V, T74K h75G7C6-3 VH- IGHV1- CDR- 133 VL- IGKHV3- CDR- 139 g3 46*01/ grafted, Q1E, g2 11*01/ grafted, I2N, JH6b R38K, M48I, JK4 A44S, Y50F, M70L, R72V, F72Y T74K h75G7C6-4 VH- IGHV1- CDR- 133 VL- IGKHV3- CDR- 140 g3 46*01/ grafted, Q1E, g3 11*01/ grafted, I2N, JH6b R38K, M48I, JK4 A44S, Y50F, M70L, R72V, I59V, F72Y T74K h75G7C6-5 VH- IGHV1- CDR- 134 VL- IGKHV3- CDR- 137 g0 46*01/ grafted, g0 11*01/ grafted, JH6b JK4 h75G7C6-6 VH- IGHV1- CDR- 134 VL- IGKHV3- CDR- 138 g0 46*01/ grafted, g1 11*01/ grafted, I2N, JH6b JK4 Y50F, F72Y h75G7C6-7 VH- IGHV1- CDR- 134 VL- IGKHV3- CDR- 139 g0 46*01/ grafted, g2 11*01/ grafted, I2N, JH6b JK4 A44S, Y50F, F72Y h75G7C6-8 VH- IGHV1- CDR- 134 VL- IGKHV3- CDR- 140 g0 46*01/ grafted, g3 11*01/ grafted, I2N, JH6b JK4 A44S, Y50F, I59V, F72Y h75G7C6-9 VH- IGHV1- CDR- 135 VL- IGKHV3- CDR- 137 g1 46*01/ grafted, M48I, g0 11*01/ grafted, JH6b M70L, R72V JK4 h75G7C6-10 VH- IGHV1- CDR- 135 VL- IGKHV3- CDR- 138 g1 46*01/ grafted, M48I, g1 11*01/ grafted, I2N, JH6b M70L, R72V JK4 Y50F, F72Y h75G7C6-11 VH- IGHV1- CDR- 135 VL- IGKHV3- CDR- 139 g1 46*01/ grafted, M48I, g2 11*01/ grafted, I2N, JH6b M70L, R72V JK4 A44S, Y50F, F72Y h75G7C6-12 VH- IGHV1- CDR- 135 VL- IGKHV3- CDR- 140 g1 46*01/ grafted, M48I, g3 11*01/ grafted, I2N, JH6b M70L, R72V JK4 A44S, Y50F, I59V, F72Y h75G7C6-13 VH- IGHV1- CDR- 136 VL- IGKHV3- CDR- 137 g2 46*01/ grafted, R38K, g0 11*01/ grafted, JH6b M48I, M70L, JK4 R72V, T74K h75G7C6-14 VH- IGHV1- CDR- 136 VL- IGKHV3- CDR- 138 g2 46*01/ grafted, R38K, g1 11*01/ grafted, I2N, JH6b M48I, M70L, JK4 Y50F, F72Y R72V, T74K h75G7C6-15 VH- IGHV1- CDR- 136 VL- IGKHV3- CDR- 139 g2 46*01/ grafted, R38K, g2 11*01/ grafted, I2N, JH6b M48I, M70L, JK4 A44S, Y50F, R72V, T74K F72Y h75G7C6-16 VH- IGHV1- CDR- 136 VL- IGKHV3- CDR- 140 g2 46*01/ grafted, R38K, g3 11*01/ grafted, I2N, JH6b M48I, M70L, JK4 A44S, Y50F, R72V, T74K I59V, F72Y
TABLE-US-00031 TABLE 27 Human Human germline germline antibody Heavy antibody Light heavy chain Donor chain light chain Donor chain variable framework variable variable framework variable Heavy region residues region Light region residues region Antibody chain template and back SEQ ID chain template and back SEQ ID number name VH mutations NO. name Vk mutations NO. c63A10A7 VH None CDR 9 VL None CDR 13 h63A10A7-17 VH- IGHV1- CDR- 143 VL- IGKV1- CDR- 149 g4 46*01/JH4 grafted, T30I, g4 39*01/JK2 grafted, Q3L, R38K, A40R, Y36L, A43S, M48I, R67K, P44S, L46A, V68A, M70L, T69A, F71Y A97T h63A10A7-18 VH- IGHV1- CDR- 144 VL- IGKV1- CDR- 149 g5 46*01/JH4 grafted, T30I, g4 39*01/JK2 grafted, Q3L, R38K, A40R, Y36L, A43S, M48I, R67K, P44S, L46A, V68A, M70L, T69A, F71Y R72A, T74Q, V79A, A97T h63A10A7-19 VH- IGHV1- CDR- 145 VL- IGKV1- CDR- 149 g6 46*01/JH4 grafted, T30I, g4 39*01/JK2 grafted, Q3L, R38K, A40R, Y36L, A43S, R67K, V68A, P44S, L46A, A97T T69A, F71Y h63A10A7-20 VH- IGHV1- CDR- 146 VL- IGKV1- CDR- 149 g7 46*01/JH4 grafted, T30I, g4 39*01/JK2 grafted, Q3L, M48I, R67K, Y36L, A43S, V68A, M70L, P44S, L46A, A97T T69A, F71Y h63A10A7-21 VH- IGHV1- CDR- 147 VL- IGKV1- CDR- 149 g8 46*01/JH4 grafted, T30I, g4 39*01/JK2 grafted, Q3L, R38K, A40R, Y36L, A43S, M48I, M70L, P44S, L46A, A97T T69A, F71Y h63A10A7-22 VH- IGHV1- CDR- 143 VL- IGKV1- CDR- 148 g4 46*01/JH4 grafted, T30I, g3 39*01/JK2 grafted, Y36L, R38K, A40R, A43S, P44S, M48I, R67K, L46A, T69A, V68A, M70L, F71Y A97T h63A10A7-23 VH- IGHV1- CDR- 143 VL- IGKV1- CDR- 150 g4 46*01/JH4 grafted, T30I, g5 39*01/JK2 grafted, Q3L, R38K, A40R, Y36L, L46A, M48I, R67K, T69A, F71Y V68A, M70L, A97T h63A10A7-24 VH- IGHV1- CDR- 143 VL- IGKV1- CDR- 151 g4 46*01/JH4 grafted, T30I, g6 39*01/JK2 grafted, Q3L, R38K, A40R, Y36L, A43S, M48I, R67K, P44S, L46A, V68A, M70L, F71Y A97T h63A10A7-25 VH- IGHV1- CDR- 143 VL- IGKV1- CDR- 152 g4 46*01/JH4 grafted, T30I, g7 39*01/JK2 grafted, Q3L, R38K, A40R, A43S, P44S, M48I, R67K, L46A, T69A, V68A, M70L, F71Y A97T h63A10A7-26 VH- IGHV1- CDR- 143 VL- IGKV1- CDR- 153 g4 46*01/JH4 grafted, T30I, g8 39*01/JK2 grafted, Q3L, R38K, A40R, A43S, P44S, M48I, R67K, L46A, T69A V68A, M70L, A97T h63A10A7-27 VH- IGHV1- CDR- 141 VL- IGKV1- CDR- 149 g2 46*01/JH4 grafted, T30I, g4 39*01/JK2 grafted, Q3L, A97T Y36L, A43S, P44S, L46A, T69A, F71Y h63A10A7-28 VH- IGHV1- CDR- 141 VL- IGKV1- CDR- 150 g2 46*01/JH4 grafted, T30I, g5 39*01/JK2 grafted, Q3L, A97T Y36L, L46A, T69A, F71Y h63A10A7-29 VH- IGHV1- CDR- 141 VL- IGKV1- CDR- 151 g2 46*01/JH4 grafted, T30I, g6 39*01/JK2 grafted, Q3L, A97T Y36L, A43S, P44S, L46A, F71Y h63A10A7-30 VH- IGHV1- CDR- 141 VL- IGKV1- CDR- 152 g2 46*01/JH4 grafted, T30I, g7 39*01/JK2 grafted, Q3L, A97T A43S, P44S, L46A, T69A, F71Y h63A10A7-31 VH- IGHV1- CDR- 141 VL- IGKV1- CDR- 153 g2 46*01/JH4 grafted, T30I, g8 39*01/JK2 grafted, Q3L, A97T A43S, P44S, L46A, T69A h63A10A7-32 VH- IGHV1- CDR- 142 VL- IGKV1- CDR- 149 g3 46*01/JH4 grafted, T30I, g4 39*01/JK2 grafted, Q3L, M48I, M70L, Y36L, A43S, A97T P44S, L46A, T69A, F71Y h63A10A7-33 VH- IGHV1- CDR- 142 VL- IGKV1- CDR- 150 g3 46*01/JH4 grafted, T30I, g5 39*01/JK2 grafted, Q3L, M48I, M70L, Y36L, L46A, A97T T69A, F71Y h63A10A7-34 VH- IGHV1- CDR- 142 VL- IGKV1- CDR- 151 g3 46*01/JH4 grafted, T30I, g6 39*01/JK2 grafted, Q3L, M48I, M70L, Y36L, A43S, A97T P44S, L46A, F71Y h63A10A7-35 VH- IGHV1- CDR- 142 VL- IGKV1- CDR- 152 g3 46*01/JH4 grafted, T30I, g7 39*01/JK2 grafted, Q3L, M48I, M70L, A43S, P44S, A97T L46A, T69A, F71Y h63A10A7-36 VH- IGHV1- CDR- 142 VL- IGKV1- CDR- 153 g3 46*01/JH4 grafted, T30I, g8 39*01/JK2 grafted, Q3L, M48I, M70L, A43S, P44S, A97T L46A, T69A h63A10A7-37 VH- IGHV1- CDR- 145 VL- IGKV1- CDR- 148 g6 46*01/JH4 grafted, T30I, g3 39*01/JK2 grafted, Y36L, R38K, A40R, A43S, P44S, R67K, V68A, L46A, T69A, A97T F71Y h63A10A7-38 VH- IGHV1- CDR- 146 VL- IGKV1- CDR- 148 g7 46*01/JH4 grafted, T30I, g3 39*01/JK2 grafted, Y36L, M48I, R67K, A43S, P44S, V68A, M70L, L46A, T69A, A97T F71Y h63A10A7-39 VH- IGHV1- CDR- 147 VL- IGKV1- CDR- 148 g8 46*01/JH4 grafted, T30I, g3 39*01/JK2 grafted, Y36L, R38K, A40R, A43S, P44S, M48I, M70L, L46A, T69A, A97T F71Y Note: The meaning of in the table is and, which refers to a parallel relationship.
[0284] cDNAs (i.e., the sequences respectively shown in SEQ ID NO. 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173 and 174 in the sequence listing) are synthesized based on the amino acid sequence of the light chain variable region and heavy chain variable region of each humanized antibody; the heavy chain cDNA is digested with FspAI and AfeI, and the light chain cDNA is digested with FspAI and BsiwI; then the cDNA fragments are inserted into an expression vector containing a signal peptide and a human heavy chain antibody IgG1 constant region and an expression vector containing a signal peptide and a human antibody light chain kappa constant region (wherein the expression vectors are purchased from Invitrogen, and the recombination steps are also completed by Shanghai ChemPartner Co., Ltd) through FspAI/AfeI or FspAI/BsiwI restriction enzyme sites, respectively, thereby obtaining recombinant plasmids which are verified by sequencing; an alkaline lysis kit (purchased from MACHEREY-NAGEL) is used for a medium-amount extraction of the recombinant plasmids having a high purity, wherein the recombinant plasmids have a mass of at least 500 g and filtered through a 0.22 m filter membrane (purchased from Millopore) for transfection.
[0285] Before transfection, 293E cells (purchased from Invitrogen) are cultured in a Freestyle 293 expression medium (purchased from Invitrogen). During transfection, the Freestyle 293 expression medium is added with 10% (v/v) F68 (purchased from Invitrogen) to a final F68 concentration of 0.1% (v/v), thereby obtaining a Freestyle 293 expression medium containing 0.1% (v/v) F68 (i.e., a medium A). 5 mL of the medium A and 200 g/mL PEI (purchased from Sigma) are mixed evenly to obtain a medium B. 5 mL of the medium A and 100 g of the heavy and light chain recombinant plasmids (the mass ratio of the heavy chain recombinant plasmid to the light chain recombinant plasmid is in the range of 1:1-1:3) are mixed evenly to obtain a medium C. After 5 minutes, the medium B and the medium C are combined and mixed evenly, and allowed to stand for 15 minutes to obtain a mixed solution D. 10 mL of the mixed solution D was slowly added to 100 mL of the Freestyle 293 expression medium containing 293E cells until the cell density of 293E is 1.510.sup.6 cells/mL, wherein the addition was performed with shaking to avoid excessive concentration of PEI, and the mixture was placed in a shaker for culture, with the shaker being set to 37 C., 130 RPM and 8% CO.sub.2 (v/v). The next day peptone is added to a final concentration of 0.5% (w/v). On days 5 to 7, the antibody titer of the culture broth is measured. On days 6 and 7, the supernatant is collected by centrifugation (3500 RPM, 30 minutes) and filtered through a 0.22 m filter membrane to obtain the filtered cell supernatant for purification.
[0286] During antibody purification, the continuously produced endotoxin-free chromatography columns and protein A fillers (purchased from GE) are rinsed with 5 column volumes of 0.5 M NaOH. Then 5 column volumes of PBS (a PBS buffer, pH 7.4) are used for equilibration to neutrality, and then the filtered cell supernatant is loaded onto the column, and the flow-through fluid is collected if necessary. After loaded onto the column, 5-fold column volumes of PBS are used for washing. 5-fold column volumes of 0.1 M Glycine-HCl (pH 3.0) is used for eluting, and the eluate is collected, and immediately added with 0.1-fold volume of 1 M Tris-HCl (1.5 M NaCl) (pH 8.5) to neutralize the EGFRvIII antibody. All the solutions used above need to be freshly prepared. The EGFRvIII antibody is harvested, and then dialyzed in 1PBS for 4 hours to avoid endotoxin contamination. After dialysis, the concentration is measured using a spectrophotometer or a kit; the purity of the antibodies is measured using HPLC-SEC; and the endotoxin content of the antibodies is detected using an endotoxin detection kit (purchased from Lonza). The obtained EGFRvIII antibody is subjected to characterization (the operation steps are as described in example 13 below).
Example 14 Identification of Humanized Anti-EGFRvIII Antibody
A. Binding of the Antibody to an EGFRvIII Protein Detected by an Enzyme-Linked Immunosorbent Assay (ELISA)
[0287] The purified humanized anti-EGFRvIII antibody obtained in example 12 is reacted with a human EGFRvIII-hFc protein.
[0288] The purified immunogen A (EGFRvIII-hFc) obtained in example 1 (for the preparation method, see step (I) in example 1) is diluted with PBS to a final concentration of 1.0 g/mL, and then added to a 96-well ELISA plate at 100 l/well. The plate is sealed with a plastic film and incubated at 4 C. overnight. The next day, the plate is washed twice with a plate washing solution [PBS+0.01% (v/v) Tween20], and added with a blocking solution [PBS+0.01% (v/v) Tween20+1% (w/w) BSA] for blocking 2 hours at room temperature. The blocking solution is discarded, and the purified EGFRvIII chimeric antibody obtained in example 2 is added at 100 l/well. The plate is incubated for 2 hours at 37 C., and then washed 3 times with the plate washing solution [PBS+0.01% (v/v) Tween20]. The plate is added with an HRP (horseradish peroxidase) labeled secondary antibody (purchased from Sigma), incubated at 37 C. for 2 hours, and then washed 3 times with the plate washing solution [PBS+0.01% (v/v) Tween20]. The plate is added with 100 l of TMB substrate/well, incubated for 30 minutes at room temperature, and then added with 100 l of stopping solution (1.0N HCl)/well. A450 nm value is read using an ELISA plate reader (SpectraMax 384plus, purchased from Molecular Device). The results are shown in
TABLE-US-00032 TABLE 28 Binding reaction of humanized h75G7C6 antibody mutant with human EGFRvIII-hFc protein detected by ELISA Maximum Antibody name EC50 (unit: nM) OD450 value Negative control antibody None 0.17 Chimeric antibody 0.50 2.50 c75G7C6 Humanized antibody 0.49 3.41 h75G7C6-l Humanized antibody 0.38 3.38 h75G7C6-2 Humanized antibody 0.51 3.44 h75G7C6-3 Humanized antibody 0.45 3.37 h75G7C6-4 Humanized antibody 0.70 3.32 h75G7C6-5 Humanized antibody 0.68 3.20 h75G7C6-6 Humanized antibody 1.10 2.97 h75G7C6-7 Humanized antibody 2.00 2.72 h75G7C6-8 Humanized antibody 0.58 2.83 h75G7C6-9 Humanized antibody 0.66 2.98 h75G7C6-10 Humanized antibody 0.67 2.96 h75G7C6-11 Humanized antibody 0.57 2.73 h75G7C6-12 Humanized antibody 0.48 3.16 h75G7C6-13 Humanized antibody 0.32 3.03 h75G7C6-14 Humanized antibody 0.29 3.19 h75G7C6-15 Humanized antibody 0.27 2.97 h75G7C6-16
TABLE-US-00033 TABLE 29 Binding reaction of humanized h63A10A7 antibody mutant with human EGFRvIII-hFc protein detected by ELISA Maximum Antibody name EC50 (unit: nM) OD450 value Negative control antibody None 0.1 Chimeric antibody 0.22 3.6 c63A10A7 Humanized antibody 0.01 3.71 h63A10A7-17 Humanized antibody 0.01 3.71 h63A10A7-18 Humanized antibody 0.17 3.63 h63A10A7-19 Humanized antibody 0.18 3.55 h63A10A7-20 Humanized antibody 0.17 3.60 h63A10A7-21 Humanized antibody 0.15 3.57 h63A10A7-22 Humanized antibody 0.33 3.47 h63A10A7-23 Humanized antibody 0.15 3.67 h63A10A7-24 Humanized antibody 0.17 3.61 h63A10A7-25 Humanized antibody 0.16 3.65 h63A10A7-26 Humanized antibody 0.31 3.5 h63A10A7-27 Humanized antibody 1.73 2.8 h63A10A7-28 Humanized antibody 0.33 3.4 h63A10A7-29 Humanized antibody 0.26 3.5 h63A10A7-30 Humanized antibody 0.30 3.4 h63A10A7-31 Humanized antibody 0.21 3.5 h63A10A7-32 Humanized antibody 0.72 3.2 h63A10A7-33 Humanized antibody 0.27 3.5 h63A10A7-34 Humanized antibody 0.18 3.4 h63A10A7-35 Humanized antibody 0.21 3.3 h63A10A7-36 Humanized antibody 0.28 3.6 h63A10A7-37 Humanized antibody 0.22 3.7 h63A10A7-38 Humanized antibody 0.24 3.6 h63A10A7-39
B. Binding of Antibody to EGFRvIII Expressing Cell Detected by Fluorescence Activated Cell Sorting (FACS)
[0289] The preparation of U87MG-EGFRvIII required for FACS detection is as described in the preparation of the immunogen B in example 1. Normal human primary hepatocytes are purchased from BioreclamationIVT, and A431 tumor cells are purchased from ATCC. The recovered hepatocytes are directly used for FACS detection, and the recovered U87MG-EGFRvIII and A431 cells are subjected to an expanded culture to 90% confluence in a T-75 cell culture flask; the medium is sucked out, and the cells are washed twice with a HBSS buffer (Hanks Balanced Salt Solution) (purchased from Invitrogen), and then treated with an enzyme-free cell dissociation solution (Versene solution: purchased from Life Technology) and collected. The cells are washed twice with the HBSS buffer and are counted, and then the cells are diluted with the HBSS buffer to 210.sup.6 cells/ml, added with 1% goat serum blocking solution, with the percentage being the mass percentage, and incubated on ice for 30 minutes, and then centrifugally washed twice with the HBSS buffer. The collected cells are resuspended to 210.sup.6 cells/mL with an FACS buffer (HBSS+2% FBS, with the percentage being the volume percentage), added to a 96-well FACS reaction plate at 100 L/well, then added with 100 L/well of the purified EGFRvIII antibody (samples to be tested) obtained in example 12, and incubated on ice for 2 hours. The plate is centrifugally washed twice with the FACS buffer, added with 100 L/well of a 1:1000 diluted fluorescence (Alexa 488) labeled secondary antibody (purchased from Invitrogen), and incubated on ice for 1 hour. The plate is centrifugally washed 3 times with the FACS buffer, added with 100 L/well of a fixing solution [4% (v/v) paraformaldehyde] to resuspend the cells, and centrifugally washed twice with the FACS buffer after 10 minutes. The cells are resuspended in 100 L of the FACS buffer, and the results are detected and analyzed by FACS (FACS Calibur, purchased from BD). Data analysis is performed by using the software (CellQuest) to obtain the mean fluorescence intensity (MFI) of the cells. Data is further analyzed by using the software (GraphPad Prism5) to perform data fitting and calculate EC50.
[0290] The analysis results are as shown in tables 30 and 31, and
TABLE-US-00034 TABLE 30 Binding reaction of humanized h75G7C6 antibody variant with EGFRvIII on the surface of U87MG-hEGFRvIII cells detected by FACS U87MG-EGFRvIII Maximum mean EC50 (unit: Antibody name fluorescence intensity nM) Negative control hIgG 0 None Chimeric antibody 18060 5.75 c75G7C6 Humanized antibody 18267 4.93 h75G7C6-1 Humanized antibody 18306 5.67 h75G7C6-2 Humanized antibody 18594 5.60 h75G7C6-3 Humanized antibody 18686 5.57 h75G7C6-4 Humanized antibody 17772 6.07 h75G7C6-5 Humanized antibody 17110 5.96 h75G7C6-6 Humanized antibody 17203 5.74 h75G7C6-7 Humanized antibody 17593 11.62 h75G7C6-8 Humanized antibody 18232 5.83 h75G7C6-9 Humanized antibody 18039 5.32 h75G7C6-10 Humanized antibody 17646 5.11 h75G7C6-11 Humanized antibody 17791 5.20 h75G7C6-12 Humanized antibody 18719 6.77 h75G7C6-13 Humanized antibody 18473 6.37 h75G7C6-14 Humanized antibody 18856 6.45 h75G7C6-15 Humanized antibody 18872 6.74 h75G7C6-16
TABLE-US-00035 TABLE 31 Binding reaction of humanized h63A10A7 antibody variant with EGFRvIII on the surface of U87MG- hEGFRvIII cells detected by FACS U87MG-EGFRvIII Maximum mean fluorescence EC50 (unit: Antibody name intensity nM) Negative control hIgG 0 None Chimeric antibody 11650 3.80 c63A10A7 Humanized antibody 21123 3.27 h63A10A7-19 Humanized antibody 22590 1.56 h63A10A7-20 Humanized antibody 23511 2.34 h63A10A7-21 Humanized antibody 23519 1.58 h63A10A7-22 Humanized antibody 20151 4.62 h63A10A7-23 Humanized antibody 24165 1.83 h63A10A7-24 Humanized antibody 24676 1.32 h63A10A7-25 Humanized antibody 23692 1.79 h63A10A7-26 Humanized antibody 9969 7.68 h63A10A7-27 Humanized antibody 6691 43.18 h63A10A7-28 Humanized antibody 9783 8.55 h63A10A7-29 Humanized antibody 9542 4.86 h63A10A7-30 Humanized antibody 8977 5.72 h63A10A7-31 Humanized antibody 10416 4.66 h63A10A7-32 Humanized antibody 8258 17.98 h63A10A7-33 Humanized antibody 10137 5.08 h63A10A7-34 Humanized antibody 10088 3.52 h63A10A7-35 Humanized antibody 9871 5.20 h63A10A7-36 Humanized antibody 10213 5.35 h63A10A7-37 Humanized antibody 10739 3.03 h63A10A7-38 Humanized antibody 10651 4.10 h63A10A7-39
[0291] In addition, humanized 75G7C6 and 63A10A7 antibody variants can also weakly bind to the wild-type EGFR. A431 cells (human epidermal cell cancer cell line) overexpress a large amount of wild-type EGFR proteins, and normal human primary hepatocytes also have a certain amount of wild-type EGFR protein expressed on the surface thereof; as shown in
[0292] Similarly, each variant of the humanized 63A10A7 antibody has a different binding ability regarding the EGFR protein on the surfaces of A431 and normal human primary hepatocytes. As shown in
TABLE-US-00036 TABLE 32 Binding reaction of humanized h75G7C6 antibody variant with EGFR on the surfaces of A431 tumor cells and normal human primary hepatocytes detected by FACS A431 tumor Normal primary Selective window Antibody name cells hepatocytes (MFI.sub.A431/Hepatocytes) Negative control hIgG 388 219 1.8 Anti-EGFR positive 23918 433 55.2 control antibody Chimeric antibody 8394 317 26.5 c75G7C6 Humanized antibody 9780 277 35.5 h75G7C6-1 Humanized antibody 11751 250 47.0 h75G7C6-2 Humanized antibody 10717 238 45.0 h75G7C6-9 Humanized antibody 11451 256 44.7 h75G7C6-13 Humanized antibody 11452 248 46.2 h75G7C6-14 Humanized antibody 13002 287 45.3 h75G7C6-15 Humanized antibody 12433 269 46.2 h75G7C6-16
TABLE-US-00037 TABLE 33 Binding reaction of humanized h63A10A7 antibody variant with EGFR on the surfaces of A431 tumor cells and normal human primary hepatocytes detected by FACS A431 tumor Normal primary Selective window Antibody name cells hepatocytes (MFI.sub.A431/Hepatocytes) Negative control hIgG 388 219 2.0 Anti-EGFR positive 23918 433 55.2 control antibody Chimeric antibody 4729 299 15.8 c63A10A7 Humanized antibody 10550 677 15.6 h63A10A7-17 Humanized antibody 9137 553 16.5 h63A10A7-18 Humanized antibody 3832 439 8.7 h63A10A7-19 Humanized antibody 7462 486 15.4 h63A10A7-20 Humanized antibody 7927 568 14.0 h63A10A7-21 Humanized antibody 9379 592 15.8 h63A10A7-22 Humanized antibody 3999 661 6.0 h63A10A7-23 Humanized antibody 9861 502 19.6 h63A10A7-24 Humanized antibody 7360 433 17.0 h63A10A7-25 Humanized antibody 6131 435 14.1 h63A10A7-26 Humanized antibody 209 262 0.8 h63A10A7-27 Humanized antibody 87 268 0.3 h63A10A7-28 Humanized antibody 212 262 0.8 h63A10A7-29 Humanized antibody 126 260 0.5 h63A10A7-30 Humanized antibody 112 254 0.4 h63A10A7-31 Humanized antibody 626 267 2.3 h63A10A7-32 Humanized antibody 92 252 0.4 h63A10A7-33 Humanized antibody 624 254 2.5 h63A10A7-34 Humanized antibody 236 253 0.9 h63A10A7-35 Humanized antibody 160 245 0.7 h63A10A7-36 Humanized antibody 1321 265 5.0 h63A10A7-37 Humanized antibody 1061 254 4.2 h63A10A7-38 Humanized antibody 1955 261 7.5 h63A10A7-39
C. Detection of Binding Affinity of Humanized Anti-EGFRvIII Antibody
[0293] In order to evaluate the binding specificity and affinity of the humanized anti-EGFRvIII antibody to the EGFRvIII and EGFR proteins, on an OctetRED (purchased from Pall) instrument, an anti-human Fc biosensor AHC (purchased from ForteBio) is used to bind the humanized h75G7C6 antibody variant to be tested, which then binds to the EGFRvIII-his (purchased from Sino Biological) or EGFR-his (purchased from Sino Biological) proteins, and detected by means of bio-layer interferometry (BLI) technology. BLI technology involves: detecting the interaction of the molecules fixed on the sensor with the molecules in the solution, which results in that the biofilm thickness is increased, and the interference spectrum curve is shifted along the direction of increased wavelength, wherein the phase shift of light waves can be detected by workstation in real time, and analyzed to quantitatively obtain the changes in the number of molecules on the sensor surface and related concentration and kinetic data. The binding rate (K.sub.a), dissociation rate (K.sub.dis) and binding affinity (K.sub.D) of the humanized h75G7C6 antibody variant to the EGFRvIII and EGFR proteins are as shown in tables 34 and 35, wherein the chimeric antibody c75G7C6 is used as a control.
TABLE-US-00038 TABLE 34 Binding affinity of humanized h75G7C6 antibody variant to EGFRvIII protein Antibody name Ka (1/Ms) Kdis (1/s) KD (M) Chimeric antibody 9.14E+04 1.32E04 1.45E09 c75G7C6 Humanized antibody 8.28E+04 1.41E04 1.71E09 h75G7C6-1 Humanized antibody 9.06E+04 1.40E04 1.54E09 h75G7C6-2 Humanized antibody 9.89E+04 1.75E04 1.77E09 h75G7C6-9 Humanized antibody 1.25E+05 1.81E04 1.45E09 h75G7C6-13
TABLE-US-00039 TABLE 35 Binding affinity of humanized h75G7C6 antibody variant to EGFR protein Antibody name Ka (1/Ms) Kdis (1/s) KD (M) Chimeric antibody 1.65E+04 3.62E03 2.20E07 c75G7C6 Humanized antibody 1.72E+04 3.53E03 2.05E07 h75G7C6-1 Humanized antibody 2.03E+04 4.05E03 2.00E07 h75G7C6-2 Humanized antibody 2.02E+04 4.35E03 2.15E07 h75G7C6-9 Humanized antibody 2.25E+04 4.76E03 2.11E07 h75G7C6-13
[0294] OctetRED results show that the binding affinity to the EGFRvIII and EGFR proteins of the humanized h75G7C6 antibody variant is very close to that of the chimeric antibody c75G7C6, which verifies that the humanized h75G7C6 antibody does not significantly reduce the antigen binding activity compared to the chimeric antibody. Furthermore, the binding affinity of the humanized h75G7C6 antibody variant to the EGFRvIII protein is about 1.5 nM, and the binding affinity thereof to the EGFR protein is about 0.2 uM.
[0295] Although the specific embodiments of the present invention have been described above, it will be understood by those skilled in the art that these are merely illustrative, and that various alterations or modifications can be made to these embodiments without departing from the principle and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.