CN113698492B - Human mesothelin chimeric antigen receptor and application thereof - Google Patents

Human mesothelin chimeric antigen receptor and application thereof Download PDF

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CN113698492B
CN113698492B CN202110557515.1A CN202110557515A CN113698492B CN 113698492 B CN113698492 B CN 113698492B CN 202110557515 A CN202110557515 A CN 202110557515A CN 113698492 B CN113698492 B CN 113698492B
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CN113698492A (en
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胡齐悦
童建松
陶维康
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Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
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Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • A61K39/001168—Mesothelin [MSLN]
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Abstract

The present disclosure relates to human mesothelin chimeric antigen receptors and uses thereof. In particular, the disclosure relates to Chimeric Antigen Receptors (CARs) containing specific mesothelin binding domains, T cells expressing the chimeric antigen receptors, and their use for treating diseases associated with mesothelin expression.

Description

Human mesothelin chimeric antigen receptor and application thereof
Technical Field
The present disclosure relates to antibodies that specifically bind to mesothelin, T cells expressing Chimeric Antigen Receptors (CARs) that specifically bind to mesothelin, and uses thereof for treating diseases associated with the expression of mesothelin.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Chimeric Antigen Receptor (CAR) is a molecule comprising the following three basic units: (1) an extracellular antigen binding motif, (2) a linking/transmembrane motif, and (3) an intracellular T cell signaling motif, which is specific for the selected antigen. Targeted T cell therapies using CARs have achieved clinical success in the treatment of some hematological malignancies, however, using CAR-expressing T cell therapies for solid tumor treatment remains difficult.
Mesothelin (MSLN) is a 40kDa cell surface Glycosyl Phosphatidylinositol (GPI) -linked glycoprotein. The protein was synthesized as a 70kD precursor and then proteolytically processed. The 30KD amino terminus of mesothelin is secreted and is known as Megakaryocyte Potentiator (MPF) (Yamaguchi et al, J.biol. Chem.269:805808, 1994). The carboxy terminus of 40kD remains bound to the membrane as mature mesothelin (Chang et al, natl. Acad. Sci. USA93:136140, 1996).
MSLN is not necessary in normal tissues, is present in very few normal adult tissues including mesothelium, but is abundantly expressed abnormally in, for example, mesothelioma, pancreatic and ovarian cancers (Chang K, pastan I.molecular cloning of mesothelin, a differentiation antigenpresent on mesothelium, mesothelioma, and ovarian cancer. Proc Natl Acad SciUSA 1996; 93:136-40.). Excessive mesothelin expression was first found in mesothelioma and ovarian Cancer, and then in lung, esophagus, pancreas, stomach, biliary tract, endometrium, thymus, colon, breast Cancer in succession (Morello A, sarelain M, adeumlili P.Mesothelin-Targeted CARs: driving T cellsto Solid Tumors [ J ]. Cancer Discovery,2015,6 (2)). 90% of the epithelioid malignant pleural mesothelioma, 69% of lung adenocarcinoma, 60% of breast Cancer, 46% of esophageal carcinoma express mesothelin (Morello A, sarelain M, adusumilli PS.Mesothelin-Targeted CARs: driving T cells to Solid Tumors [ J ]. Cancer discover, 2015,6 (2)). Mesothelin is also expressed in more aggressive lung, breast (triple negative) and esophageal cancers (highly dysplastic and adenocarcinoma). Therefore, mesothelin is taken as a tumor antigen and is a good target of CAR-T.
Therefore, the development of the CAR-T aiming at MSLN is continued, and the method has important significance for improving the tumor treatment effect. Currently, CART for MSLN is disclosed in the following patents, such as WO2013063419, CN109097396, WO2017112741, WO2020043152, etc.
Disclosure of Invention
The present disclosure provides chimeric antigen receptors for MSLN (MSLN-CARs) with novel sequences that have been experimentally validated to find that these MSLN-CARs can specifically bind to human MSLN and can effectively kill tumor target cells in vivo and in vitro.
In some embodiments, the Chimeric Antigen Receptor (CAR) molecule comprises:
i) An anti-mesothelin binding domain,
ii) a transmembrane domain, and
iii) An intracellular signaling domain;
wherein the anti-mesothelin binding domain comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an LCDR as shown below:
LCDR1 is as X 1 ASQRISSYLS (SEQ ID NO: 94);
LCDR2 such as X 2 ASX 3 LX 4 S (SEQ ID NO: 95);
LCDR3 is shown as QSYAYFDSNNWHA (SEQ ID NO: 6); and
the heavy chain variable region comprises HCDR as shown below:
HCDR1 such as FYX 5 YAC (SEQ ID NO: 91);
HCDR2 such as CIYTAGGGSSTYYAX 6 X 7 X 8 KG (SEQ ID NO: 92);
HCDR3 such as STX 9 NTRSTYYLNX 10 (SEQ ID NO: 93);
wherein X is 1 Selected from Q or R, X 2 Selected from A or G, X 3 Selected from S or T, X 4 Selected from A or Q, X 5 Selected from F, Y or H, X 6 Selected from S or D, X 7 Selected from W or S, X 8 Selected from A or V, X 9 Selected from S or A, X 10 Selected from T or L.
In some embodiments, a CAR molecule as described previously, wherein the mesothelin binding domain comprises a light chain variable region and a heavy chain variable region, wherein:
a) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:14 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
b) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
c) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
d) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:14 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:11, HCDR1, HCDR2 and HCDR3;
e) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:11, HCDR1, HCDR2 and HCDR3;
f) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:11, HCDR1, HCDR2 and HCDR3;
g) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:11, HCDR1, HCDR2 and HCDR3;
h) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:14 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:12, HCDR1, HCDR2 and HCDR3;
i) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:12, HCDR1, HCDR2 and HCDR3;
j) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:12, HCDR1, HCDR2 and HCDR3;
k) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:12, HCDR1, HCDR2 and HCDR3;
l) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:14 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
m) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
n) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
o) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
p) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:14 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
q) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
r) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
s) said light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:9 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
t) said light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:9 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
u) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:9 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
v) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:10 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3; or (b)
w) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:10 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3.
In some embodiments, a CAR molecule as described previously, wherein the mesothelin binding domain comprises a light chain variable region and a heavy chain variable region, wherein:
the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:12, HCDR1, HCDR2 and HCDR3; or (b)
The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3.
In some embodiments, a CAR as described previously, the anti-mesothelin binding domain thereof comprises a light chain variable region and a heavy chain variable region, wherein:
the sequence of the light chain variable region is shown in SEQ ID NO: 25. 26, 27, 28, 29, 30 or 32; and/or
The heavy chain variable region sequence is shown in SEQ ID NO: 16. 17, 18, 19, 20, 21, 22, 23, 24, 31 or 33.
In some embodiments, a CAR as described previously, the anti-mesothelin binding domain thereof comprises a light chain variable region and a heavy chain variable region, wherein:
a1 The light chain variable region sequence is shown as SEQ ID NO:25 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and
the heavy chain variable region sequence is shown in SEQ ID NO: 16. 19, 20, 21 or 22, or a sequence corresponding to SEQ ID NO: 16. 19, 20, 21 or 22, respectively, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity;
b1 The light chain variable region sequence is shown as SEQ ID NO:26, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and
The heavy chain variable region sequence is shown in SEQ ID NO: 16. 18, 19, 20, 21, 22, 23, or 33, or a sequence corresponding to SEQ ID NO: 16. 18, 19, 20, 21, 22, 23, or 33, respectively, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity;
c1 The light chain variable region sequence is shown as SEQ ID NO:27, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and
the heavy chain variable region sequence is shown in SEQ ID NO: 16. 18, 19, 20, 21, 22 or 23, or a sequence which hybridizes to SEQ ID NO: 16. 18, 19, 20, 21, 22, or 23, respectively, have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity;
d1 The light chain variable region sequence is shown as SEQ ID NO:28, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and
the heavy chain variable region sequence is shown in SEQ ID NO: 16. 18, 19, 20, 21, 22 or 23, or a sequence which hybridizes to SEQ ID NO: 16. 18, 19, 20, 21, 22, or 23, respectively, have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity;
e1 The light chain variable region sequence is shown as SEQ ID NO:29, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and
the heavy chain variable region sequence is shown in SEQ ID NO: 16. 18, 19, 20, 21, 22, 23, or 24, or a sequence corresponding to SEQ ID NO: 16. 18, 19, 20, 21, 22, 23, or 24, respectively, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity;
f1 The light chain variable region sequence is shown as SEQ ID NO:30, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and
the heavy chain variable region sequence is shown in SEQ ID NO: 19. 20, 21, 23 or 24, or a sequence which hybridizes to SEQ ID NO: 19. 20, 21, 23 or 24, respectively, have at least 95% sequence identity; or (b)
g1 The light chain variable region sequence is shown as SEQ ID NO:32, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and
the heavy chain variable region sequence is shown in SEQ ID NO:31, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.
In some embodiments, a CAR as described previously, the anti-mesothelin binding domain thereof comprises the amino acid sequence as set forth in SEQ ID NO:29 and a light chain variable region as set forth in SEQ ID NO:20, and a heavy chain variable region shown in seq id no; or (b)
Comprising the amino acid sequence as set forth in SEQ ID NO:30 and the light chain variable region as set forth in SEQ ID NO: 21.
In some embodiments, a CAR as described previously, the anti-mesothelin binding domain thereof is an scFv.
In some embodiments, a CAR as described previously, the anti-mesothelin binding domain scFv of which comprises:
as set forth in SEQ ID NO: 25. 26, 27, 28, 29, 30 or 32, and/or
As set forth in SEQ ID NO: 16. 17, 18, 19, 20, 21, 22, 23, 24, 31 or 33.
In some embodiments, a CAR as described previously, the anti-mesothelin binding domain scFv of which comprises:
as set forth in SEQ ID NO:29 and a light chain variable region as set forth in SEQ ID NO:20, and a heavy chain variable region shown in seq id no; or (b)
As set forth in SEQ ID NO:30 and the light chain variable region as set forth in SEQ ID NO: 21.
In some embodiments, a CAR as described previously, wherein the anti-mesothelin binding domain comprises the amino acid sequence as set forth in SEQ ID NO: 34-74.
In some embodiments, a CAR as described previously, wherein the anti-mesothelin binding domain comprises the amino acid sequence as set forth in SEQ ID NO: 51. 52, 53, 58, 59 or 60.
In some embodiments, a CAR as described previously, wherein the transmembrane domain comprises a transmembrane domain selected from any one of the following proteins: the α, β or ζ chain of T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
In some embodiments, a CAR as described previously, wherein the transmembrane domain comprises SEQ ID NO: 77.
In some embodiments, a CAR as described above, wherein the anti-mesothelin binding domain is linked to the transmembrane domain by a hinge region.
In some embodiments, a CAR as described previously, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 76.
In some embodiments, a CAR as described previously, wherein the intracellular signaling domain is a functional signaling domain of cd3ζ;
in some embodiments, a CAR as described previously, wherein said functional signaling domain of cd3ζ comprises a sequence as set forth in SEQ ID NO: 79.
In some embodiments, a CAR as described previously, wherein the intracellular signaling domain further comprises a co-stimulatory domain.
In some embodiments, a CAR as described previously, wherein the co-stimulatory domain comprises a functional signaling domain selected from any one of the following proteins: OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), CD278 (also known as "ICOS") and 4-1BB (CD 137).
In some embodiments, the CAR as described previously, wherein the co-stimulatory domain is a functional signaling domain of 4-1 BB.
In some embodiments, a CAR as described previously, wherein the functional signaling domain of 4-1BB comprises the amino acid sequence as set forth in SEQ ID NO: 78.
In some embodiments, a CAR as described previously, wherein the intracellular signaling domain comprises the amino acid sequence as set forth in SEQ id no:78 and/or the sequence shown as SEQ ID NO: 79.
In some embodiments, the CAR as described previously, further comprises a leader sequence.
In some embodiments, a CAR as described previously, wherein the leader sequence comprises the sequence set forth in SEQ ID NO: 75.
In some embodiments, a CAR as described previously, comprising a sequence as set forth in SEQ ID NO: 80-87.
In some embodiments, a CAR as described previously, comprising a sequence as set forth in SEQ ID NO:82 or 85.
The present disclosure also provides an antibody that specifically binds to mesothelin comprising an anti-mesothelin binding domain as described in any one of the preceding claims.
In some embodiments, wherein the antibody that specifically binds mesothelin comprises a light chain variable region and a heavy chain variable region, wherein:
the light chain variable region comprises an LCDR as shown below:
LCDR1 is as X 1 ASQRISSYLS (SEQ ID NO: 94);
LCDR2 such as X 2 ASX 3 LX 4 S (SEQ ID NO: 95);
LCDR3 is shown as QSYAYFDSNNWHA (SEQ ID NO: 6); and
the heavy chain variable region comprises HCDR as shown below:
HCDR1 such as FYX 5 YAC (SEQ ID NO: 91);
HCDR2 such as CIYTAGGGSSTYYAX 6 X 7 X 8 KG (SEQ ID NO: 92);
HCDR3 such as STX 9 NTRSTYYLNX 10 (SEQ ID NO: 93);
wherein X is 1 Selected from Q or R, X 2 Selected from A or G, X 3 Selected from S or T, X 4 Selected from A or Q, X 5 Selected from F, Y or H, X 6 Selected from S or D, X 7 Selected from W or S, X 8 Selected from A or V, X 9 Selected from S or A, X 10 Selected from T or L.
In some embodiments, an antibody that specifically binds mesothelin as described previously, comprising a light chain variable region and a heavy chain variable region as set forth in any one of the following, wherein:
a) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:14 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
b) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
c) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:14 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:11, HCDR1, HCDR2 and HCDR3;
d) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:11, HCDR1, HCDR2 and HCDR3;
e) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:11, HCDR1, HCDR2 and HCDR3;
f) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:11, HCDR1, HCDR2 and HCDR3;
g) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:14 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:12, HCDR1, HCDR2 and HCDR3;
h) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:12, HCDR1, HCDR2 and HCDR3;
i) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:12, HCDR1, HCDR2 and HCDR3;
j) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:12, HCDR1, HCDR2 and HCDR3;
k) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:14 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
l) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
m) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
n) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
o) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:14 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
p) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
q) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
r) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:9 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
s) said light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:9 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
t) said light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:9 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3;
u) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:10 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3; or (b)
v) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:10 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3.
In some embodiments, an antibody that specifically binds to mesothelin as described previously, wherein the antibody that specifically binds mesothelin comprises:
A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:5 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:12, HCDR1, HCDR2 and HCDR3; or (b)
A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3.
In some embodiments, an antibody that specifically binds mesothelin as described previously, comprising a light chain variable region and a heavy chain variable region, wherein:
the sequence of the light chain variable region is shown in SEQ ID NO: 25. 26, 27, 28, 29 or 30; and/or
The heavy chain variable region sequence is shown in SEQ ID NO: 16. 17, 18, 19, 20, 21, 22, 23 or 24.
In some embodiments, an antibody that specifically binds mesothelin as described previously, comprising a light chain variable region and a heavy chain variable region, wherein:
a1 The light chain variable region sequence is shown as SEQ ID NO:25, or at least 95% sequence identity thereto; and
the heavy chain variable region sequence is shown in SEQ ID NO: 16. 19, 20, 21 or 22, or a sequence corresponding to SEQ ID NO: 16. 19, 20, 21 or 22, respectively, have at least 95% sequence identity;
b1 The light chain variable region sequence is shown as SEQ ID NO:26 or at least 95% sequence identity thereto; and
the heavy chain variable region sequence is shown in SEQ ID NO: 16. 18, 19, 20, 21, 22 or 23, or a sequence which hybridizes to SEQ ID NO: 16. 18, 19, 20, 21, 22, or 23 has at least 95%, 96%, 97%, 98%, or 99% sequence identity;
c1 The light chain variable region sequence is shown as SEQ ID NO:27, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; and
the heavy chain variable region sequence is shown in SEQ ID NO: 16. 18, 19, 20, 21, 22 or 23, or a sequence which hybridizes to SEQ ID NO: 16. 18, 19, 20, 21, 22, or 23 has at least 95%, 96%, 97%, 98%, or 99% sequence identity;
d1 The light chain variable region sequence is shown as SEQ ID NO:28, or at least 95% sequence identity thereto; and
the heavy chain variable region sequence is shown in SEQ ID NO: 16. 18, 19, 20, 21, 22 or 23, or a sequence which hybridizes to SEQ ID NO: 16. 18, 19, 20, 21, 22, or 23 has at least 95%, 96%, 97%, 98%, or 99% sequence identity;
e1 The light chain variable region sequence is shown as SEQ ID NO:29, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; and
The heavy chain variable region sequence is shown in SEQ ID NO: 16. 18, 19, 20, 21, 22, 23, or 24, or a sequence corresponding to SEQ ID NO: 16. 18, 19, 20, 21, 22, 23, or 24 has at least 95%, 96%, 97%, 98%, or 99% sequence identity; or (b)
f1 The light chain variable region sequence is shown as SEQ ID NO:30, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; and
the heavy chain variable region sequence is shown in SEQ ID NO: 19. 20, 21, 23 or 24, or a sequence which hybridizes to SEQ ID NO: 19. 20, 21, 23, or 24 has at least 95%, 96%, 97%, 98%, or 99% sequence identity.
In some embodiments, an antibody that specifically binds mesothelin as described previously, comprising a light chain variable region and a heavy chain variable region, wherein:
the sequence of the light chain variable region is shown in SEQ ID NO:29 and the heavy chain variable region sequence shown in SEQ ID NO: shown at 20; or (b)
The sequence of the light chain variable region is shown in SEQ ID NO:30 and the heavy chain variable region sequence shown in SEQ ID NO: 21.
In some embodiments, the antibody that specifically binds mesothelin as described previously is a single chain antibody.
In some embodiments, a single chain antibody that specifically binds mesothelin as described previously comprises:
As set forth in SEQ ID NO: 16. 17, 18, 19, 20, 21, 22, 23 or 24, and/or
As set forth in SEQ ID NO: 25. 26, 27, 28, 29 or 30.
In some embodiments, an antibody that specifically binds mesothelin as described previously, comprising an amino acid sequence as set forth in SEQ ID NO: 34-72.
In some embodiments, an antibody that specifically binds mesothelin as described previously, comprising an amino acid sequence as set forth in SEQ ID NO: 51. 52, 53, 58, 59 or 60.
The present disclosure also provides a nucleic acid molecule encoding any of the foregoing CARs or an antibody that specifically binds mesothelin of any of the foregoing.
In some embodiments, wherein the nucleic acid molecule comprises a sequence as set forth in SEQ ID NO:88 or 89.
The present disclosure also provides a recombinant vector comprising the nucleic acid molecule of any one of the preceding claims.
In some embodiments, wherein the recombinant vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, or retroviral vector.
In some embodiments, wherein the recombinant vector further comprises a promoter.
In some embodiments, wherein the promoter is an EF-1 promoter.
In some embodiments, wherein the EF-1 promoter comprises the sequence of SEQ ID NO: 90.
The present disclosure also provides a cell comprising a recombinant vector as described above.
In some embodiments, wherein the cell is an immune effector cell and expresses the CAR of any one of the preceding claims.
In some embodiments, wherein the immune effector cell is an NK cell, a macrophage, a dendritic cell, or a T cell.
In some embodiments, wherein the immune effector cell is a cd8+ T cell or a cd4+ T cell.
The present disclosure also provides a method of producing a cell comprising the step of transducing the aforementioned recombinant vector into an immune effector cell.
The present disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of an antibody that specifically binds mesothelin, or a nucleic acid molecule, or a cell, as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.
The present disclosure also provides the use of a cell expressing a CAR as defined in any one of the preceding claims, or an antibody as defined above that specifically binds mesothelin, or a nucleic acid molecule as defined above, or a pharmaceutical composition as defined above, in the manufacture of a medicament for the treatment or prevention of cancer; preferably, wherein the cancer is selected from: mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, cholangiocarcinoma, breast cancer, malignant pleural mesothelioma, non-small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic ductal adenoma, ovarian cancer, colorectal cancer, and bladder cancer; more preferably, wherein the cancer is associated with mesothelin expression.
In some embodiments, the disclosure provides a method of treating and cancer comprising administering to a subject a therapeutically effective amount of a cell expressing an isolated CAR of any one of the preceding claims, or an antibody that specifically binds mesothelin as described previously, or a nucleic acid molecule as described previously, or a pharmaceutical composition as described previously; preferably, wherein the cancer is selected from: mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, cholangiocarcinoma, breast cancer, malignant pleural mesothelioma, non-small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic ductal adenoma, ovarian cancer, colorectal cancer, and bladder cancer; more preferably, wherein the cancer is associated with mesothelin expression.
In some embodiments, the disclosure provides a cell expressing an isolated CAR of any one of the preceding claims, or an antibody that specifically binds mesothelin, or a nucleic acid molecule, or a pharmaceutical composition, as described above, for use in treating cancer; preferably, wherein the cancer is selected from: mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, cholangiocarcinoma, breast cancer, malignant pleural mesothelioma, non-small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic ductal adenoma, ovarian cancer, colorectal cancer, and bladder cancer; more preferably, wherein the cancer is associated with mesothelin expression.
Description of the drawings:
fig. 1: different MSLN-scFv antibody cell levels bound to human MSLN experimental results.
Fig. 2: MSLN-CAR mediated T cell activation assay results.
Fig. 3: FIG. 3A shows MSLN-CART cell viability versus time curves; FIG. 3B shows a plot of fold proliferation versus time for MSLN-CART cells; FIG. 3C shows the CAR positive rate in MSLN-CART cells; FIG. 3D shows the MFI of MSLN-CART cells.
Fig. 4: FIG. 4A shows MSLN-CART cell proliferation curves without antigen repetitive stimulation; FIG. 4B shows the proliferation curve of MSLN-CART cells measured after antigen re-stimulation.
Fig. 5: shows the killing rate of different MSLN-CART cells on target cells.
Fig. 6: FIG. 6A shows IL-2 secretion during killing of target cells by MSLN-CART cells; FIG. 6B shows secretion of IFNγ during killing of target cells by MSLN-CART cells.
Fig. 7: FIG. 7A shows the change in mouse body weight; fig. 7B shows the change in tumor volume in mice.
Fig. 8: continuous proliferation of MSLN-CART cells in mice.
Detailed Description
Definition of the definition
For easier understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
The term "chimeric antigen receptor" or "CAR" refers to a group of polypeptides that, when in an immune effector cell, provide the cell with specificity for a target cell (typically a cancer cell) and have intracellular signaling. In certain embodiments, the CAR comprises at least one extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), including functional signaling domains derived from stimulatory molecules and/or co-stimulatory molecules as defined below. In certain aspects, the sets of polypeptides are contiguous with each other. In certain embodiments, the set of polypeptides includes a dimerization switch that allows the polypeptides to be coupled to each other in the presence of the dimerization molecule, e.g., the antigen binding domain may be coupled to an intracellular signaling domain. In one aspect, the stimulatory molecule is a zeta chain that binds to the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from a costimulatory molecule. In one aspect, the costimulatory molecule is selected from the costimulatory molecules described herein, such as 4-1BB (i.e., CD 137), CD27, and/or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino terminus (N-terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., scFv) during cellular processing and localization of the CAR to a cell membrane.
The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell to regulate the activity of the cell via a defined signaling pathway by either generating a second messenger or by responding to such a messenger as an effector.
The term "mesothelin" refers to a 40kDa protein mesothelin that is anchored to the cell membrane by a 31kDa shed fragment (known as megakaryocyte potentiator, MPF) that is bounded by Glycosyl Phosphatidylinositol (GPI) bonds and the amino terminus. Both fragments contain an N-glycosylation site. The term also refers to a soluble splice variant of the 40kDa carboxy-terminal fragment, also known as "soluble mesothelin/MPF-related". Preferably, the term refers to the human mesothelin of GenBank accession No. AAH03512.1 and naturally cleaved portions thereof, e.g., expressed on a cell membrane (e.g., a cancer cell membrane).
The "antibody" as described in the present disclosure refers to an immunoglobulin, and the complete full-length antibody is a tetrapeptide chain structure formed by connecting two heavy chains and two light chains through inter-chain disulfide bonds. The immunoglobulin heavy chain constant region has different amino acid compositions and sequences, and also has different antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or isotypes of immunoglobulins, igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α, and epsilon chains, respectively. The same class of Ig can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, e.g., igG can be classified into IgG1, igG2, igG3, and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each of the five classes of Ig may have either a kappa chain or a lambda chain.
The sequences of the heavy and light chains of antibodies, near the N-terminus, vary widely, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions, arranged in the order from amino-to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.
Antibodies of the disclosure include human antibodies.
The term "murine antibody" is in the present disclosure a monoclonal antibody against human MSLN prepared according to the knowledge and skill in the art. The preparation is performed by injecting the test subjects with MSLN antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In a preferred embodiment of the present disclosure, the murine anti-MSLN antibody may further comprise a murine kappa, lambda chain or variant light chain constant region, or further comprise a murine IgG1, igG2, igG3 or variant heavy chain constant region.
The term "chimeric antibody (chimeric antibody)" refers to an antibody in which the variable region of an antibody of a certain species (e.g., murine) is fused to the constant region of an antibody of another species (e.g., human) to reduce the immune response induced by a heterologous antibody. For example, a chimeric antibody is established by first establishing a hybridoma secreting a murine specific monoclonal antibody, then cloning a variable region gene from murine hybridoma cells, then cloning a human antibody constant region gene as desired, linking the murine variable region gene with the human constant region gene to form a chimeric gene, then inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic or prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the MSLN chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the MSLN chimeric antibody further comprises a heavy chain constant region of a human IgG1, igG2, igG3, igG4 or variant thereof, preferably a human IgG1, igG2 or IgG4 heavy chain constant region, or an IgG1, igG2 or IgG4 variant using amino acid mutations (e.g., L234A and/or L235A mutations, and/or S228P mutations).
The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting the CDR sequences of an antibody of a certain species (e.g., murine) into the framework of the variable regions of a human antibody, i.e., into the framework sequences of a different type of human germline antibody. The heterologous reaction induced by chimeric antibodies due to the large number of carrying heterologous protein components can be overcome. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. Germline DNA sequences for human heavy and light chain variable region genes can be found, for example, in the "VBase" human germline sequence database (available in Internet www.mrccpe.com.ac.uk/VBase) and in Kabat, E.A. et al, 1991, sequences of Proteins of Immunological Interest, 5 th edition. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity. Humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation mutations of the CDRs by yeast display.
The terms "human antibody" and "human antibody" are used interchangeably to refer to a human immunoglobulin sequence from which one or more variable and constant regions are derived. One preferred mode is where all the variable and constant regions are derived from human immunoglobulin sequences, i.e. "fully human antibodies" or "fully human antibodies". These antibodies can be prepared in a variety of ways, including isolation of B cells from human PBMC, spleen, lymph node tissue by phage display techniques, construction of a natural single-chain phage human antibody library, or screening of the obtained antibodies by immunization of transgenic mice that can express human antibody light and heavy chains. The human antibodies of the present disclosure also include antibodies obtained by mutation of one or more amino acids on the basis of the human antibodies that still bind to the antigen of interest.
The "antibodies" of the present disclosure include antigen binding fragments capable of binding an antigen in addition to full length antibodies. The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution) with an epitope of an antigen. Examples of antibody fragments include, but are not limited to, fab ', F (ab') 2, fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments (e.g., bivalent fragments comprising two Fab fragments linked at the hinge region by disulfide bonds), and isolated CDRs or other epitope-binding fragments of antibodies. Antigen binding fragments may also be incorporated into single domain antibodies, maxiantibodies, miniantibodies, nanobodies, intracellular antibodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFv (see, e.g., hollinger and Hudson, nature Biotechnology 23:1126-1136,2005). The antigen binding fragment may also be grafted onto a polypeptide-based scaffold, such as fibronectin type III (Fn 3) (see U.S. Pat. No. 6,703,199, which describes a fibronectin polypeptide miniantibody).
The term "scFv" or "single chain antibody" refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, an scFv may have the VL and VH variable regions described in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.
The CAR portion of the present disclosure comprising an antibody or antibody fragment thereof can exist in a variety of forms, wherein the antigen binding domain is expressed as part of a continuous polypeptide chain, including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) humanized antibody, or a bispecific antibody (Harlow et al, 1999,In:Using Antibodies:ALaboratory Manual,Cold Spring Harbor Laboratory Press,NY;Harlow et al, 1989,In:Antibodies:A Laboratory Manual,Cold Spring Harbor,New York;Houston et al, 1988,Proc.Natl.Acad.Sci.USA 85:5879-5883; bird et al, 1988,Science 242:423-426). In one aspect, the antigen binding domain of the CAR compositions of the present disclosure comprises an antibody fragment. In another aspect, the CAR comprises an antibody fragment comprising an scFv.
The term "complementarity determining region", "CDR" or "hypervariable region" refers to one of the 6 hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding. Typically, there are three CDRs (HCDR 1, HCDR2, HCDR 3) in each heavy chain variable region, and three CDRs (LCDR 1, LCDR2, LCDR 3) in each light chain variable region. The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes, including "Kabat" numbering convention (see Kabat et Al (1991), "Sequences of Proteins of Immunological Interest", 5 th edition, public Health Service, national Institutes of Health, bethesda, MD), "Chothia" numbering convention (see Al-Lazikani et Al, (1997) JMB 273:927-948) and ImMunoGenTics (IMGT) numbering convention (Lefranc M.P.), immunolist, 7, 132-136 (1999); lefranc, M.P. et Al, dev. Comp. Immunol.,27, 55-77 (2003) et Al, for example, for classical format, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR 1), 50-65 (HCDR 2) and 95-102 (HCDR 3), the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-35 (HCDR 1), 50-65 (VL 2) and 95 (HCDR 3), the amino acid residues in the light chain variable domain (VL) are numbered 24-35 (VL) and 50-35 (VL 2), the amino acid residues in the light chain variable domain (VL) are numbered 26-35 (VL) and 95-35 (VL) and the amino acid residues in the amino acid variable domain (VL) are numbered 26-35 (VL) and 95-35 (HCDR 2) and 95 (VL) are combined by amino acid numbers 50-35, VL 2 and VL 2-35 (VL 1) and VL 2 (VL 1) are defined by amino acid (HCDR 1) and HCDR2 50-56 (LCDR 2) and 89-97 (LCDR 3). Following the IMGT rules, the CDR amino acid residues in VH are approximately 26-35 (CDR 1), 51-57 (CDR 2) and 93-102 (CDR 3), and the CDR amino acid residues in VL are approximately 27-32 (CDR 1), 50-52 (CDR 2) and 89-97 (CDR 3). Following IMGT rules, CDR regions of antibodies can be determined using the procedure IMGT/DomainGap alignment. Unless otherwise indicated, the antibody variable region and CDR sequences referred to in the examples of the present disclosure are all subject to the "Kabat" numbering convention.
The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen.
The term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
Amino acid sequence "identity" refers to the percentage of amino acid residues in a first sequence that are identical to amino acid residues in a second sequence, with gaps introduced between the aligned amino acid sequences and where necessary to achieve the maximum percentage of sequence identity, and without any conservative substitutions being considered part of the sequence identity. For the purpose of determining the percent amino acid sequence identity, the alignment may be accomplished in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN-2 or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences compared.
Methods for producing and purifying antibodies and antigen binding fragments are well known in the art, such as the guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15. For example, mice may be immunized with human EpCAM or a fragment thereof, the resulting antibodies can be renatured, purified, and amino acid sequencing can be performed using conventional methods. Antigen binding fragments can likewise be prepared by conventional methods. The antibodies or antigen binding fragments of the invention are engineered to incorporate one or more human FR regions in the CDR regions of non-human origin. Human FR germline sequences can be obtained from the website http:// IMGT. Cines. FR of ImMunoGeneTics (IMGT), or from the journal of immunoglobulins, 2001ISBN012441351 by aligning IMGT human antibody variable region germline gene databases with MOE software.
The engineered antibodies or antigen binding fragments of the disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site of the Fc region. Stable clones were obtained by expressing antibodies that specifically bound to human EpCAM. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing conditioned buffer. Non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.
The term "cancer" refers to a disease characterized by uncontrolled growth of abnormal cells. Cancer cells may locally spread or pass through the blood stream and lymphatic system to other parts of the body. Examples of various cancers are described herein, including but not limited to mesothelioma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.
The term "disease associated with the expression of mesothelin" includes, but is not limited to, diseases associated with the expression of mesothelin or disorders associated with cells expressing mesothelin, including, for example, proliferative diseases such as cancer or malignant tumor or pre-cancerous disorders such as mesothelial hyperplasia; or a non-cancer related indication associated with cells expressing mesothelin. Examples of various cancers that express mesothelin include, but are not limited to, mesothelioma, lung cancer, ovarian cancer, pancreatic cancer, and the like.
The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced into the antibodies or antibody fragments of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the present disclosure may be replaced with an amino acid residue from the same side chain family, and altered CARs, e.g., binding to mesothelin, may be tested using the functional assays described herein.
The term "stimulation" refers to the primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex or CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signaling event such as, but not limited to, signaling via the TCR/CD3 complex or signaling via a signaling domain of a suitable NK receptor or CAR. Stimulation may mediate altered expression of certain molecules.
The term "stimulatory molecule" refers to a sequence molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides cytoplasmic signaling that modulates in a stimulatory manner the activation of an immune cell for at least some aspects of the immune cell signaling pathway. In one aspect, the signal is a primary signal initiated by, for example, binding of the TCR/CD3 complex to a peptide-loaded MHC molecule, and which results in mediating T cell responses including, but not limited to, proliferation, activation, differentiation, and the like. The primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing cytoplasmic signaling sequences particularly useful in the present disclosure include, but are not limited to, those derived from: cd3ζ, common fcrγ (FCER 1G), fcγriia, fcrβ (FcEpsilon R1 b), cd3γ, cd3δ, cd3ε, CD79a, CD79b, DAP10, and DAP12. In the specific CARs of the disclosure, the intracellular signaling domain in any one or more CARs of the disclosure comprises an intracellular signaling sequence, such as a primary signaling sequence of CD3- ζ. In the specific CARs of the disclosure, the primary signaling sequence of CD3- ζ is as set forth in SEQ ID NO:79 or equivalent residues from a non-human species such as mice, rodents, monkeys, apes, etc.
An "immune effector cell" refers to a cell that is involved in an immune response (e.g., promotes an immune effector response). Examples of immune effector cells include T cells, such as alpha/beta T cells and gamma/delta T cells, B cells, natural Killer (NK) cells, natural Killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.
The term "antigen presenting cell" or "APC" refers to an immune system cell, such as a helper cell (e.g., B-cell, dendritic cell, etc.), that presents foreign antigens complexed with a Major Histocompatibility Complex (MHC) on its surface. T-cells can recognize these complexes using their T-cell receptor (TCR). APCs process antigens and present them to T-cells.
The term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain produces a signal that promotes immune effector function of a CAR-containing cell, e.g., a CART cell. Examples of immune effector functions in e.g. CART cells include cell lysis activity and helper activity, including secretion of cytokines.
In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may comprise a co-stimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signaling or antigen-independent stimulation. For example, in CART, the primary intracellular signaling domain may comprise a cytoplasmic sequence of a T cell receptor, and the co-stimulatory intracellular signaling domain may comprise a cytoplasmic sequence from a co-receptor or co-stimulatory molecule. The primary intracellular signaling domain may include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of first order cytoplasmic signaling sequences containing ITAM include, but are not limited to, those derived from: cd3ζ, common fcrγ (FCER 1G), fcγriia, fcrβ (FcEpsilon R1 b), cd3γ, cd3δ, cd3ε, CD79a, CD79b, DAP10, and DAP12.
The term "ζ" or alternatively "ζ chain", "CD3- ζ" or "TCR ζ" is defined as a protein as provided by genbank acc.no. bag36664.1 or an equivalent residue from a non-human species (e.g. mouse, rodent, monkey, ape, etc.), and "ζ stimulating domain" or "CD3- ζ stimulating domain" or "TCR- ζ stimulating domain" is defined as an amino acid residue from the cytoplasmic domain of the ζ chain or a functional derivative thereof sufficient to functionally transmit the initiation signal required for T cell activation. In one aspect, the cytoplasmic domain of ζ comprises residues 52 to 164 of GenBank acc.no. bag36664.1 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) as a functional homolog thereof.
The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that promote an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278) and 4-1BB (CD 137). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF 1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2 Rbeta, IL2 Rgamma, IL7 Ralpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD, ITGAL, CD11a, LFA-1, ITGAM, CD11B, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD 226), SLAMF4 (CD 244, 2B 4), CD84, CD96 (Tactive), CEACAM1, CRTAM, ly9 (CD 229), CD160 (BY 55), PSGL1, CD100 (SEMA 4D), CD69, SLAMF6 (NTB-A, ly 108), SLAM (SLAMF 1, CD150, IPO-3), BLAME (SLAMF 8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, and ligands that specifically bind CD 83.
The co-stimulatory intracellular signaling domain may be the intracellular portion of a co-stimulatory molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), and NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD 137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, antigen-1 associated with lymphocyte function (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, SLAMF, NKp80, CD160, B7-H3, and ligands that specifically bind CD83, and the like.
The intracellular signaling domain may comprise all intracellular portions of the molecule or all native intracellular signaling domains, or functional fragments or derivatives thereof.
The term "4-1BB" refers to a member of the TNFR superfamily having an amino acid sequence as provided by GenBank Acc.No. AAA62478.2, or an equivalent residue from a non-human species such as mouse, rodent, monkey, ape, or the like. In one aspect, the "4-1BB co-stimulatory domain" is defined as amino acid residues 214-255 of GenBank Acc.No. AAA62478.2, or equivalent residues from a non-human species such as mouse, rodent, monkey, ape, etc.
The term "encode" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, in a biological process as a template for the synthesis of other polymers and macromolecules having defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and biological properties derived therefrom. Thus, a gene, cDNA or RNA encodes a protein if transcription and translation of mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is normally provided in the sequence listing, and the non-coding strand, which serves as a template for transcription of a gene or cDNA, may be referred to as a protein or other product encoding the gene or cDNA.
Unless otherwise indicated, "a nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding a protein may contain introns in some forms.
The term "effective amount" or "therapeutically effective amount" is used interchangeably herein and refers to the amount of a compound, formulation, substance, or composition that is effective to achieve a particular biological result as described herein.
The term "endogenous" refers to any substance that is derived from or produced within an organism, cell, tissue or system.
The term "exogenous" refers to any substance introduced or produced from outside an organism, cell, tissue, or system.
The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
The term "transfer vector" refers to a composition of matter that includes an isolated nucleic acid and that can be used to deliver the isolated nucleic acid into the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes autonomously replicating plasmids or viruses. The term should also be construed to further include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus (lentiviral) vectors, and the like.
The term "expression vector" refers to a vector comprising a recombinant polynucleotide that comprises an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporated into the recombinant polynucleotide.
The term "lentivirus" refers to a family of retroviruses. Lentiviruses are unique among retroviruses in that they are capable of infecting non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, so they are one of the most efficient methods of gene delivery vehicles. HIV, SIV and FIV are all examples of lentiviruses. The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, and specifically includes self-inactivating lentiviral vectors, as provided in Milone et al mol. Ther.17 (8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used in the clinic include, but are not limited to, LENTIVECTOR gene delivery techniques such as from Oxford BioMedica, vector systems from Lentigen LENTIMAX, and the like. Non-clinical species of lentiviral vectors are also available and will be known to those skilled in the art.
The term "isolated" refers to a change or removal from a natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide, partially or completely isolated from coexisting materials in its natural state, is "isolated. The isolated nucleic acid or protein may be present in a substantially purified form, or may be present in a non-natural environment such as, for example, a host cell.
The term "operative linkage" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous to each other and must be in the same reading frame when binding two protein coding regions.
The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. The protein or peptide must contain at least two amino acids and there is no limit to the maximum number of amino acids that can comprise the sequence of the protein or peptide. Polypeptides include any peptide or protein comprising two or more amino acids bound to each other by peptide bonds. As used herein, the term refers to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) as well as longer chains (which are also commonly referred to in the art as proteins, which are of various types). "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
The term "promoter" refers to a DNA sequence recognized by, or introduced into, a synthetic mechanism of a cell that is required to initiate specific transcription of a polynucleotide sequence.
The term "flexible polypeptide linker" refers to a peptide linker composed of amino acids such as glycine and/or serine residues used alone or in combination that links together variable heavy and variable light chain regions. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises an amino acid sequence (Gly 4 Ser) n Wherein n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3.n=4, n=5, and n=6, n=7, n=8, n=9, and n=10. Also included within the scope of the present disclosure are the linkers described in WO2012/138475, which is incorporated herein by reference.
The term "transfected" or "transformed" or "transduced" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. The cells include primary subject cells and their progeny.
"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not.
"pharmaceutical composition" means a mixture containing one or more antibodies that specifically bind MSLN, CART cells, and other chemical components, such as physiological/pharmaceutically acceptable carriers and excipients, as described herein. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.
The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivery of an antibody or antigen-binding fragment. The carrier may be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (e.g., antioxidant, antimicrobial or antifungal), sweetener, absorption delaying agent, wetting agent, emulsifier, buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents, such as sugars, polyols, sorbitol, and sodium chloride.
"administering," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administration," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration," "administration," and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
The term "preventing" refers to the prophylactic or protective treatment of a disease or condition.
The term "treatment (treat, treatment, treating)" refers to slowing or ameliorating the progression, severity and/or duration of a proliferative disorder, or ameliorating one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder as a result of administration of one or more therapies (e.g., one or more therapeutic agents such as CARs of the invention). In certain embodiments, the term "treatment" refers to amelioration of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, not necessarily discernible to the patient. In other embodiments, the term "treating" refers to inhibiting the progression of a proliferative disorder physically by, for example, stabilizing a discernible symptom, physiologically by, for example, stabilizing a physical parameter, or both. In other embodiments, the term "treating" refers to reducing or stabilizing tumor size or cancer cell count.
Detailed Description
The present disclosure is further described below in conjunction with the examples, which are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not noted in the examples of the present disclosure are generally according to conventional conditions, such as an antibody technical laboratory manual for cold spring harbor, a molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.
Examples
EXAMPLE 1 preparation of anti-MSLN Single-chain antibody molecules
1. Through a large number of experiments, we constructed a series of scfvs (abbreviated as MSLN-scFv) that specifically bind MSLN, with the light/heavy chain variable region, CDR regions, full length sequences as follows:
TABLE 1 MSLN-scFv heavy chain variable region CDR
TABLE 2 MSLN-scFv light chain variable region CDR
2. The heavy and light chain variable regions of the scFv against MSLN in the present disclosure are shown below:
TABLE 3 MSLN-scFv heavy and light chain variable regions
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The VH and VL are connected through a linker, and an exemplary linker is (G4S) n, wherein n is more than or equal to 1, so as to obtain the single-chain antibody. Exemplary heavy and light chain variable regions of the antibodies described above are joined by a linker (G4S) 3 Ligation, construction of MSLN-scFv.
TABLE 4 heavy and light chain variable regions of MSLN-scFv
Note that: H1L1 represents that the heavy chain variable region of the MSLN-scFv is H1, and the light chain variable region is L1.
The sequence of an exemplary MSLN-scFv in the present disclosure is shown below:
H1L1
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：34
H1L2
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：35
H1L3：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：36
H1L4：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：37
H1L5：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：38
H3L2：
EVQLVESGGGVVQPGRSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVACIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：39
H3L3：
EVQLVESGGGVVQPGRSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVACIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：40
H3L4：
EVQLVESGGGVVQPGRSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVACIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：41
H3L5：
EVQLVESGGGVVQPGRSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVACIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：42
H4L1：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTSNTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：43
H4L2：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTSNTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：44
H4L3：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTSNTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：45
H4L4：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTSNTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：46
H4L5：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTSNTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：47
H4L6：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTSNTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：48
H5L1：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNTWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：49
H5L2：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNTWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：50
H5L3：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNTWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：51
H5L4：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNTWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：52
H5L5：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNTWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：53
H5L6：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNTWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：54
H6L1：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYYYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：55
H6L2：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYYYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：56
H6L3：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYYYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：57
H6L4：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYYYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：58
H6L5：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYYYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：59
H6L6：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYYYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：60
H7L1：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYHYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：61
H7L2：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYHYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：62
H7L3：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYHYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：63
H7L4：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYHYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：64
H7L5：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYHYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：65
H8L2：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：66
H8L3：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：67
H8L4：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：68
H8L5：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：69
H8L6：
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：70
H9L5
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：71
H9L6
EVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：72
H10L8：
EVQLVESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK
SEQ ID NO：73
H9L7：
QQQLEESGGGLVKPEGSLTLTCKASGFDLGFYFYACWVRQAPGKGLEWIACIYTAGSGSTYYASWAKGRFTISKASSTTVTLQMTSLAAADTATYFCARSTANTRSTYYLNLWGPGTLVTVSSGGGGSGGGGSGGGGSDVVMTQTPASVSEPVGGTVTIKCQASQRISSYLSWYQQKPGQRPKLLIFGASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSYAYFDSNNWHAFGGGTEVVV
SEQ ID NO：74
illustratively, the CDR region sequences of MSLN-scFv are as follows:
TABLE 5 CDR of MSLN-scFv
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The CDR regions of MSLN-scFv in the present disclosure share a commonality and can be generalized to the following formula:
the light chain variable region comprises an LCDR as shown below:
LCDR1 is as X 1 ASQRISSYLS (SEQ ID NO: 94);
LCDR2 such as X 2 ASX 3 LX 4 S (SEQ ID NO: 95);
LCDR3 is shown as QSYAYFDSNNWHA (SEQ ID NO: 6); and
the heavy chain variable region comprises HCDR as shown below:
HCDR1 such as FYX 5 YAC (SEQ ID NO: 91);
HCDR2 such as CIYTAGGGSSTYYAX 6 X 7 X 8 KG (SEQ ID NO: 92); and
HCDR3 such as STX 9 NTRSTYYLNX 10 (SEQ ID NO: 93);
wherein X is 1 Selected from Q or R, X 2 Selected from A or G, X 3 Selected from S or T, X 4 Selected from A or Q, X 5 Selected from F, Y or H, X 6 Selected from S or D, X 7 Selected from W or S, X 8 Selected from A or V, X 9 Selected from S or A, X 10 Selected from T or L.
Expression and purification of MSLN-scFv antibodies
The MSLN-scFv sequence end is added with His tag, synthesized by gene company, then cloned into Phr-IgG expression vector and expressed in Gbico Expi-CHO-S expression system. Collecting supernatant, centrifuging at high speed to remove impurities, performing affinity chromatography using Capto L (GE Heathcare 17-5478-02), washing with pure water, balancing column with PBS, binding supernatant to column, eluting target protein with 0.1M acetic acid buffer at pH3.5, and neutralizing with 1M Tris-HCl. A280 detection absorbance calculation yield, and obtaining the MSLN-scFv antibody.
EXAMPLE 2 construction of MSLN-CAR
The MSLN-scFv fragment is recombined with a transmembrane region, a signal transduction domain, a costimulatory domain and the like to construct the complete MSLN-CAR. The relevant sequences are shown below:
Cd8 leader amino acid sequence:
MALPVTALLLPLALLLHAARP
SQE ID NO：75
amino acid sequence of the cd8 hinge region:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
SQE ID NO：76
amino acid sequence of the cd8 transmembrane region:
IYIWAPLAGTCGVLLLSLVITLYC
SQE ID NO：77
4.4-1BB intracellular domain amino acid sequence:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
SQE ID NO：78
cd3 domain amino acid sequence:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SQE ID NO：79
6. the amino acid sequence of the complete MSLN-CAR was constructed as follows:
(1)H5L3-CAR：
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNTWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SQE ID NO：80
(2)H5L4-CAR：
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNTWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SQE ID NO：81
(3)H5L5-CAR：
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNTWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SQE ID NO：82
(4)H6L4-CAR：
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFDLGFYYYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQASQRISSYLSWYQQKPGKAPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYAYFDSNNWHAFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SQE ID NO：83
(5)H6L5-CAR：
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFDLGFYYYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SQE ID NO：84
(6)H6L6-CAR：
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFDLGFYYYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SQE ID NO：85
(8)H11L8-CAR：
MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SQE ID NO：86
(9)H10L7-CAR：
MALPVTALLLPLALLLHAARPQQQLEESGGGLVKPEGSLTLTCKASGFDLGFYFYACWVRQAPGKGLEWIACIYTAGSGSTYYASWAKGRFTISKASSTTVTLQMTSLAAADTATYFCARSTANTRSTYYLNLWGPGTLVTVSSGGGGSGGGGSGGGGSDVVMTQTPASVSEPVGGTVTIKCQASQRISSYLSWYQQKPGQRPKLLIFGASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSYAYFDSNNWHAFGGGTEVVVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SQE ID NO：87
(10) H5L5-CAR nucleic acid sequence:
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCAACCCGGCGGAAGCCTCAGACTGAGCTGTGCTGCCAGCGGCTTCGATCTGGGCTTCTACTTCTACGCTTGTTGGGTGAGGCAAGCCCCCGGCAAAGGACTGGAGTGGGTGAGCTGCATCTATACTGCCGGCTCCGGCTCCACATACTATGCCAGCTGGGCCAAGGGAAGGTTCACTATCTCTAGGGACAACAGCAAGAACACTCTGTATCTGCAGATGAACTCTCTGAGGGCTGAGGATACTGCCGTCTATTACTGCGCCAGAAGCACTGCCAACACAAGGAGCACTTACTATCTGAATACTTGGGGCCAAGGCACTCTGGTCACTGTGAGCAGCGGCGGAGGCGGATCAGGTGGTGGCGGATCTGGAGGTGGCGGAAGCGATATCCAGATGACTCAGTCCCCTTCCTCTCTGAGCGCTAGCGTGGGAGACAGAGTGACTATCACATGCAGGGCCAGCCAGAGGATCTCCAGCTATCTGAGCTGGTACCAGCAGAAGCCCGGCAAGGTGCCTAAGCTGCTGATCTATGGCGCCTCCACTCTGGCTAGCGGAGTGCCTTCCAGATTTAGCGGCAGCGGAAGCGGCACTGACTTCACTCTGACTATCAGCTCTCTGCAGCCAGAGGACGTGGCTACTTACTACTGCCAGAGCTACGCCTACTTCGACAGCAACAACTGGCACGCCTTTGGCGGCGGCACTAAGGTCGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTGA
SQE ID NO：88
(11) H6L6-CAR nucleic acid sequence:
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCAACCCGGCGGAAGCCTCAGACTGAGCTGTGCTGCCAGCGGCTTCGATCTGGGCTTCTACTACTACGCTTGTTGGGTGAGGCAAGCCCCCGGCAAAGGACTGGAGTGGGTGAGCTGCATCTATACTGCCGGCTCCGGCTCCACATACTATGCCAGCTGGGCCAAGGGAAGGTTCACTATCTCTAGGGACAACAGCAAGAACACTCTGTATCTGCAGATGAACTCTCTGAGGGCTGAGGATACTGCCGTCTATTACTGCGCCAGAAGCACTGCCAACACAAGGAGCACTTACTATCTGAATCTGTGGGGCCAAGGCACTCTGGTCACTGTGAGCAGCGGCGGAGGCGGATCAGGTGGTGGCGGATCTGGAGGTGGCGGAAGCGATATCCAGATGACTCAGTCCCCTTCCTCTCTGAGCGCTAGCGTGGGAGACAGAGTGACTATCACATGCCAAGCCAGCCAGAGGATCTCCAGCTATCTGAGCTGGTACCAGCAGAAGCCCGGCAAGGTGCCTAAGCTGCTGATCTATGCTGCCTCCACTCTGCAGAGCGGAGTGCCTTCCAGATTTAGCGGCAGCGGAAGCGGCACTGACTTCACTCTGACTATCAGCTCTCTGCAGCCAGAGGACGTGGCTACTTACTACTGCCAGAGCTACGCCTACTTCGACAGCAACAACTGGCACGCCTTTGGCGGCGGCACTAAGGTCGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTGA
SQE ID NO:89 the MSLN-CAR was then cloned into a lentiviral vector, producing one full length CAR molecule in a single coding frame, and expressed using the EF1 alpha promoter.
EF1 alpha promoter
ctagtggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaacgggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgaagcttcgaggggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactctacgtctttgtttcgttttctgttctgcgccgttacagatccaagctgtgaccggcgcctac
SQE ID NO:90 controls and other protein sequences used in the disclosure are as follows:
n2 (CAR against CD 19) amino acid sequence:
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITRAGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SQE ID NO：96
2.MSLN-R3-hFc4：
MNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO：97
RS7 (NC) antibody as negative control
RS7 antibody heavy chain:
QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO：98
RS7 antibody light chain:
DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO：99
EXAMPLE 3 third Generation lentiviral packaging System and lentiviral vector packaging
The third generation lentivirus packaging system used included a four plasmid system of expression plasmid, envelope plasmid and packaging plasmid for virus packaging.
Inserting the MSLN-CAR nucleic acid sequence into the multiple cloning site of pCDH-EF 1-MCS-IRES-copGGFP vector (SBI, cat#: CD 530A-2), and deleting IRES-copGGFP sequence to obtain expression plasmid pCDH-EF1 alpha-anti-MSLN-CAR; the envelope plasmid and the packaging plasmid are pMD2.G (Ubao organism, cat# VT 1443), pMDLgpRRE (Ubao organism, cat# VT 1449), and pRSV-Rev (Ubao organism, cat# VT 1445), respectively. HEK293 cells were cultured to a density of about 80% with DMEM medium plus 10% FBS, the expression plasmid pCDH-MSLN-CAR and three helper plasmids pMDLg-pRRE, pRSV-Rev, pMD2.G were mixed in a mass ratio of 4:3:2:1, HEK293 cells were transfected with PEI reagent, the supernatant was harvested 48 hours later, and the harvested lentiviral supernatant was centrifuged with 25000g high-speed centrifuge for 2 hours to obtain viruses.
EXAMPLE 4 preparation of MSLN-CART
Healthy volunteers were recruited and 100ml of blood was taken intravenously by a medical professional to anticoagulation (BD, 367886). The blood was mixed with an equal amount of PBS buffer (containing 2% fetal bovine serum). PBMC separation tube Sepmate-50 (STEMCELL Technology 86450) was taken, 15mL of Ficoll buffer (GE healthcare, 17-5442-02) was added, and then 32mL of blood-PBS mixture was added. After centrifugation at 1200g for 10min at room temperature, the upper layer of PBMC-containing suspension was rapidly poured into a clean 50mL centrifuge tube, centrifuged at 500g for 8min at room temperature, the supernatant was discarded, and the PBMC was obtained by resuspension with PBS.
Isolation kit EasySep for use TM Human T cell isolation kit (Stem cell company 17951) further isolated T cells from PBMCs. The resulting T cells were isolated at 1:1 to CD3/CD28 magnetic beads (Dynabeads) TM Human T-Activator CD3/CD28, GIBCO, 11132D), at X-VIVO 15 (Lonza, 04-418Q), containing rhIL-2 (R) at a final concentration of 100IU/mL&D, 202-IL-050) with a cell density of 1X 10 6 Individual/mL, 37 ℃,5% CO 2 Is activated under culture conditions.
After 24 hours of activation, the mixture of cells and beads was collected, the supernatant was removed by centrifugation, resuspended in the above medium (X-VIVO15+100 IU/mL rhIL-2) and removed 1X 10 6 Adding concentrated virus at MOI of 5 into 48-well plate, and adjusting cell density to 2×10 6 Centrifuge at 32℃for 1 hour at 2000g per mL; after centrifugation, the volume ratio is 1:1 supplemented with culture medium (X-VIVO15+100 IU/mL rhIL 2), placing at 37deg.C, 5% CO 2 Culturing in an incubator for 3 days. After 3 days, the beads were removed and the cell suspension was placed in the pole easy Sep TM D Magnetite Particles (STEMCELL, 19550), and standing for two minutes, removing the magnetic beads on the tube wall to obtain MSLN-CART cells. Growth was then monitored by cell counting during the culture and the MSLN-CART cells were diluted every two days with fresh medium to a cell density of 3X 10 during the culture 5 cells/mL to 2X 10 6 cells/mL。
Test case
Test example 1 anti-MSLN Single chain antibody binding experiments to target cells
hMSLN was constructed into pLVX-EF1-LucG plasmid, and after transfection of K562 cells (purchased from ATCC), the monoclonal was selected to obtain K562-hMSLN-lucG cells.
K562-hMSLN-LucG cell density was adjusted to 3X 10 6 mu.L of each well was added to each 96-well round bottom plate per mL. Washed once with PBS+2% FBS buffer, centrifuged for 500g,3 min, and the supernatant discarded. 200 μLMSLN-scFv supernatant was added to each well, incubated at room temperature for 30 minutes, centrifuged at 500g for 3 minutes, and the supernatant was discarded. Washed 2 times with PBS+2% FBS buffer, centrifuged 500g for 3 min, and the supernatant discarded. Dilution of the secondary anti-His tag antibody [ iFluor 647 ] with PBS+2% FBS buffer ](genescript, A01802-100) at a concentration of 2. Mu.g/mL, 100. Mu.L of each sample was added, incubated at room temperature for 30 minutes, centrifuged at 500g for 3 minutes, and the supernatant discarded. Wash 2 times with pbs+2% fbs buffer, centrifuge 500gx3 min, discard supernatant. 100. Mu.L PBS+2%The FBS buffer resuspended cells were air-sucked and mixed and the samples were analyzed by flow cytometry (Invitrogen, attune NxT). The flow data were analyzed by FlowJo-VX-10.4 and the geometry Mean of the fluorescence intensity of iFluor 647 was plotted and the results are shown in FIG. 1.
From the results, it can be seen that the negative control RS7 (NC) did not bind to K562-hMSLN-LucG cells, but that MSLN-scFv all bound to K562-hMSLN-LucG cells, demonstrating that the anti-MSLN single chain antibodies in the present disclosure specifically recognized human MSLN.
Test example 2 anti-MSLN Single chain antibody and antigen affinity detection
Biacore was used to determine the affinity of the MSLN-scFv single chain antibody to the antigen MSLN-R3-hFc 4.
A quantity of antigen MSLN-R3-hFc4 was affinity captured using a CM5 biosensing chip (Cat. #BR100530, GE) conjugated with anti-human antibodies, and then a concentration of anti-MSLN-scFv single chain antibodies was passed over the chip surface. The reaction signal was detected in real time using a Biacore T200 instrument to obtain binding and dissociation curves. After completion of each cycle dissociation, 3M MgCl was used 2 (human antibody Capture kit, BR-1008-39, GE) the biochip was washed and regenerated. The assay run buffer was 1 XHBS-EP buffer (GE, BR-1001-88).
The resulting data were fitted with the (1:1) Langmuir model using GE Biacore T200 Evaluation version 3.0.0 software to yield affinity values.
TABLE 6 affinity of MSLN-scFv Single chain antibodies with MSLN-R3-hFc4
The results show that the MSLN-scFv in the present disclosure has a higher affinity with MSLN-R3-hFc4 antigen.
Test example 3 MSLN-CAR mediated T cell activation ability detection
First, JNL-MSLN-CAR cells were constructed and NFAT-Lu was regulatedc Reporter T Lymphocytes (InvivoGen, cat. Jktl-nfat, abbreviated as JNL cells) has a density of 2X 10 6 mu.L/mL was added to a 96-well round bottom plate. The amount of MSLN-CAR lentivirus was calculated as moi=5, 2000g after lentivirus addition, and centrifugation at 32 ℃ for 60 minutes. Centrifuging, adding pre-heated 1640 culture medium, and adjusting cell density to 1×10 6 And (3) placing the culture medium at a volume of one mL, and culturing the culture medium in an incubator for 4 days to obtain JNL-MSLN-CAR cells.
On the fifth day, JJNL-MSLN-CAR cells and JNL-CAR-N2 cells were adjusted to a density of 1X 10 6 mu.L to 96-well flat bottom plate was used per mL, K562-hMSLN cell density was 2.5X10 5 80. Mu.L of the culture medium was placed in a 96-well flat bottom plate to which JNL-MSLN-CAR was added, and incubated in an incubator for 18 to 24 hours.
QUANTI-Luc TM Configuration of the analytical reagents: a clean 50mL centrifuge tube was taken and 25mL of sterile water was added. QUANTI-Luc TM (InvivoGen, rep-qlc 2) was dissolved by pouring all the powder into the container and stored at 4℃for one week in the absence of light.
The cells were removed from the incubator, centrifuged at 1000rpm for 5 minutes, 20. Mu.l of the culture supernatant was aspirated to 96 Kong Baiban, and 50. Mu.l of QUANTI-Luc was added TM The reagent was analyzed and mixed by shaking for 5 seconds. Immediately after mixing, the bioluminescence value was measured using an enzyme-labeled instrument, the results of which are shown in FIG. 2.
From the results, it can be seen that N2-CART cells could not be activated by K562-hMSLN, whereas JNL-MSLN-CAR cells comprising H1L3-CAR, H5L5-CAR, H6L5-CAR, H6L6-CAR and H11L8-CAR could be specifically activated by K562-hMSLN. And the extent of JNL-MSLN-CAR cell activation is related to the scFv sequence of the CAR, with JNL-MSLN-CAR cells comprising H6L6-CAR having the best effect.
Test example 4 detection of MSLN-CART cell culture Activity, proliferation Capacity and CAR expression Positive Rate
(1) And (3) detecting the CART cell culture activity rate and proliferation capacity:
MSLN-CART cells were gently mixed with a pipette, 20. Mu.L to 1.5mL centrifuge tubes were mixed with 20. Mu.L diluted Trypan Blue Solution (0.1%, gibco, 15250061), 20. Mu.L were aspirated into a cell counting plate, the cell density and cell viability were read with a cell counter, and the total number and proliferation factor of cells were calculated from the culture volume. Wherein:
Cell viability = dead cells/(live cells + dead cells) ×100%.
Fold cell proliferation = total number of viable cells on day n/total number of viable cells on day 1.
(2) Detection of positive rate of CAR expression:
MSLN-CART cell groups 5X 10 5 Centrifuging the individual cells for 3 minutes at 500g, and discarding the supernatant; 1mL of buffer (PBS+0.5% BSA) was added to the suspension, centrifuged for 500g and 3 minutes, and the supernatant was discarded and resuspended in 100. Mu.L of buffer. Adding 2 mu L of CD19 (FMC 63 antibody) primary antibody into N2-CART cells, adding 2 mu L of MSLN Fc protein into each group of MSLN-CART cells, mixing, and incubating at 4 ℃ for 30 minutes; after washing one pass with buffer, 2. Mu.L of PE sheep-anti-mouse, igG (Biolegend, 554061) and 2. Mu.L of PE anti-human Fc, igG (Biolegend, 405307) were added to each group of MSLN-CART cells and incubated at 4℃for 30 min. After incubation, the cells were resuspended by washing with buffer 2 times, adding 500. Mu.L buffer, mixing well, protected from light, and detected using a flow cytometer.
Positive rate = CAR positive cells/(CAR positive cells + CAR negative cells)
The results are shown in tables 7-9, FIGS. 3A-3D.
TABLE 7 MSLN-CART cell viability
Note that: d represents a day.
TABLE 8 multiplication factor of MSLN-CART
Note that: d represents a day.
Table 9.Car positive rate and MFI data table
Test example 5 antigen repetitive stimulation CART cell proliferation potency assay
1. First round of stimulation (days 1-4):
resuscitates target cells SKOV3 (purchased from ATCC, DMEM medium+10% fetal bovine serum, purchased from Thermo Fisher, 10566016), initial cell density 2X 10 5 And (3) culturing to logarithmic growth phase. A portion of the target cells (remaining normal culture) were resuspended in X-VIVO medium (IL-2 free, lonza, 04418Q) and plated in 48-well plates (1X 10) 5 Individual/well), placed in a 37 degree incubator, control wells were not plated with target cells. Effector cells (MSLN-CART) required for the experiment were taken, washed once with PBS, counted after resuspension of X-VIVO medium (without IL-2), and adjusted with T cells not transfected with CAR to achieve a consistent total cell mass for each group of effector cells. After target cells had attached, the supernatant was aspirated, effector cells were added to 48 well plates at a ratio of effector cells to target cells E: T ratio = 1:1, 3 duplicate wells per group, and X-VIVO medium (without IL-2) was supplemented to 600 μl/well. After 2 days of incubation of effector cells with target cells, 300. Mu. L X-VIVO medium (without IL-2) was supplemented per well. After 4 days of co-incubation, cells from the supernatant of the co-incubation system were collected, resuspended in 1 ml of X-VIVO medium (without IL-2) and counted after centrifugation, and fold expansion was calculated, wherein:
first round fold expansion = cell amount after co-incubation/initial cell amount added.
2. Second round of stimulation (days 4-8):
target cells from normal culture were resuspended in X-VIVO medium (without IL-2) and plated in 48-well plates (1X 10) 5 And 3 wells/well), placed in a 37-degree incubator (control wells without target cells), after cell attachment, effector cells were added to the 48-well plates according to the ratio of effector cells to target cells E: T ratio=1:1, 3 wells/group, and X-VIVO medium (no IL-2) was supplemented to 600 μl/well. After 2 days of incubation of effector cells with target cells, 300. Mu. l X-VIVO medium (without IL-2) was supplemented per well. After 4 days of co-incubation, the cells from the supernatant of the co-incubation were collected and centrifuged with 1 milliThe X-VIVO medium (without IL-2) was resuspended and counted, and the fold amplification was calculated, wherein:
second round fold expansion = first round fold expansion x cell amount after co-incubation/initial cell amount added
3. Third round of stimulation (days 8-12):
a third round of stimulation is performed according to the second round of stimulation method, wherein:
third round of fold expansion = first round of fold expansion x second round of fold expansion x cell amount after co-incubation/initial cell amount added.
The results of the measured CAR-T cell proliferation are shown in FIGS. 4A-4B.
From the results, the number of cells stimulated by SKOV3 cells was significantly increased compared to non-antigen stimulated MSLN-CART cells, indicating that the anti-MSLN-CART MSLN single chain antibodies were able to recognize the MSLN antigen and activate T cell expansion. In particular, H6L6-CAR, H6L4-CAR and H5L3-CAR can significantly stimulate T cell expansion.
Test example 6 detection of killing Capacity of MSLN-CART cells in vitro on target cells
After transfection of SK-OV-3 cells (purchased from ATCC) with pLVX-EF1-LucG plasmid harboring lucifrase and GFP, a monoclonal was selected to obtain SK-OV-3-LucG cell line.
Target cell (SK-OV-3-LucG) density was adjusted to 5X 10 4 mu.L of the cells were centrifuged at 600rpm for 2 minutes at 96 well flat bottom plate and incubated overnight in a cell incubator. The next day, the target cell culture medium was discarded, and 100. Mu.L of X-VIVO15 (Lonza, 04-418Q) without phenol red was added to each well. Then according to the effector cell (MSLN-CART) to target cell ratio E: t ratio= (20:1-0.625:1) effector cells were added and mixed by blowing and sucking. Centrifuge at 1000rpm for 2 minutes and incubate for 16 hours. 300g,5 min centrifugation, and supernatant discarded. The supernatant was discarded after washing with PBS. 100. Mu.L of PBS was added thereto, and 100. Mu.L of a detection solution (Promega Corp.)Luciferase Assay System, E2520), incubated at room temperature for 10-20 minutes, bioluminescence values were measured using an enzyme-labeled instrument and the kill rate calculated.
Killing = (simple target cell reading-after effector cell reading)/(simple target cell reading-medium reading) ×100%.
The killing results are shown in table 10 and fig. 5.
TABLE 10 killing Rate of MSLN-CART cells against target cells
From the results, it can be seen that MSLN-CART cells have a significant killing capacity against SK-OV-3-LucG cells, while N2 does not kill SK-OV-3-LucG cells, indicating that MSLN-CART cells can specifically kill MSLN-positive cells.
Test example 7 MSLN-CART in vitro killer target cytokine secretion
Cell supernatants after co-incubation of MSLN-CART with target cells in test 6 (E: T=1:1, E: T=5:1) were assayed for IL-2 and IFN- γ concentrations in the supernatants using human IL-2ELISA Kit II (BD, 550611) and human IFN- γ ELISA Kit II (BD, 550612), as shown in FIGS. 6A-6B.
The results show that when co-cultured with target cells, candidate cells produced large amounts of IFN-. Gamma.and IL-2 cytokines, indicating that MSLN-CART cells can all exhibit the characteristics of killer T cells.
Test example 8 evaluation of in vivo efficacy of MSLN-CART cell mouse transplantation tumor model
NDG mice (purchased by the bougainvillea corporation), females, 6-8 weeks, feeding environment: SPF stage. After one week of adaptive feeding, the mice were randomly divided into 6 groups of 6-8 mice each. Each mouse was used 5X 10 6 Subcutaneous inoculation and molding of right shoulder blade part of each SKOV3 tumor cell, and random grouping according to average tumor size after 18 days, and reinfusion of each mouse is 2.5×10 7 The body weight and the seed tumor size of the mice are recorded 2 to 3 times per week by MSLN-CART cells, and the killing condition of different CART on SK-O-V3 tumor cells in vivo is compared. The grouping of NDG mice and reinfusion of CART cells are shown in the following table:
TABLE 11 grouping of mice
Grouping Reinfusion of cell types Number of feedback cells Number of mice
1 H5L5-CAR 2.5×10 7 8
2 H6L5-CAR 2.5×10 7 8
3 H6L6-CAR 2.5×10 7 8
4 H11L8-CAR 2.5×10 7 8
5 N2 2.5×10 7 7
6 PBS Without any means for 6
The body weight and tumor size results after mice were modeled and returned to MSLN-CART cells are shown in FIGS. 7A-7B.
The results show that after reinfusion of CART cells, the body weight of the mice does not change significantly, indicating that the mice have good tolerance to CART cells. The H5L5-CAR, the H6L5-CAR and the H6L6-CAR have similar in-vivo anti-tumor effects as the H11L8-CAR, and can effectively kill SK-O-V3 tumor cells in vivo.
Test example 9. Pharmacokinetic detection of MSLN-CART cells in vivo in transplanted tumor mice
1. Proliferation assay of CART cells in mice
NDG mice (purchased by the bougainvillea corporation), females, 6-8 weeks, feeding environment: SPF stage. After one week of adaptive feeding, the mice were randomly divided into 6 groups of 7-8 mice each. Each mouse was used 5X 10 6 Subcutaneous inoculation and molding of right shoulder blade part of each SK-O-V3 tumor cell, random grouping according to average tumor size after 18 days, and reinfusion of each mouse is 2.5×10 7 And (3) MSLN-CART cells. After reinfusion of CART cells, mouse blood was collected from the orbit on days 0 (3 hours after reinfusion), 3, 7, 14, 21 and 28, and the amount of CART cells in the peripheral blood of the mice was measured, and specific groupings are shown in table 12. The number of MSLN-CART cells in the peripheral blood of mice within 4 weeks after feedback is shown in figure 8.
TABLE 12 grouping of mice
As can be seen from the results in fig. 8, after injection of MSLN-CART cells into SK-O-V3 tumor cell-modeled mice, MSLN-CART was significantly expanded in mice, but the number of N2-CART cells did not significantly proliferate, indicating that MSLN-CART cells were stimulated in mice specifically for SK-O-V3 tumor cells, thereby obtaining an expanded signal, which proliferated in large amounts.
Sequence listing
<110> Jiangsu Hengrui medicine Co., ltd
Shanghai Hengrui medicine Co., ltd
<120> human mesothelin chimeric antigen receptor and uses thereof
<160> 99
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> HCDR1
<400> 1
Phe Tyr Phe Tyr Ala Cys
1 5
<210> 2
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> HCDR2
<400> 2
Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala
1 5 10 15
Lys Gly
<210> 3
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> HCDR3
<400> 3
Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn Leu
1 5 10
<210> 4
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> LCDR1
<400> 4
Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser
1 5 10
<210> 5
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> LCDR2
<400> 5
Gly Ala Ser Thr Leu Ala Ser
1 5
<210> 6
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> LCDR3
<400> 6
Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
1 5 10
<210> 7
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> HCDR1
<400> 7
Phe Tyr Tyr Tyr Ala Cys
1 5
<210> 8
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> HCDR1
<400> 8
Phe Tyr His Tyr Ala Cys
1 5
<210> 9
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> HCDR2
<400> 9
Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Ala
1 5 10 15
Lys Gly
<210> 10
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> HCDR2
<400> 10
Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val
1 5 10 15
Lys Gly
<210> 11
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> HCDR3
<400> 11
Ser Thr Ser Asn Thr Arg Ser Thr Tyr Tyr Leu Asn Leu
1 5 10
<210> 12
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> HCDR3
<400> 12
Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn Thr
1 5 10
<210> 13
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> LCDR1
<400> 13
Arg Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser
1 5 10
<210> 14
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> LCDR2
<400> 14
Ala Ala Ser Ser Leu Gln Ser
1 5
<210> 15
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> LCDR2
<400> 15
Ala Ala Ser Thr Leu Gln Ser
1 5
<210> 16
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H1
<400> 16
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 17
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H2
<400> 17
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ala Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Thr Asn Thr
65 70 75 80
Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 18
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H3
<400> 18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ala Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 19
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H4
<400> 19
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ser Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 20
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H5
<400> 20
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 21
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H6
<400> 21
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Tyr Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 22
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H7
<400> 22
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
His Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 23
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H8
<400> 23
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 24
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H9
<400> 24
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 25
<211> 111
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> L1
<400> 25
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
85 90 95
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 26
<211> 111
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> L2
<400> 26
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
85 90 95
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 27
<211> 111
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> L3
<400> 27
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
85 90 95
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 28
<211> 111
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> L4
<400> 28
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
85 90 95
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 29
<211> 111
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> L5
<400> 29
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
85 90 95
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 30
<211> 111
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> L6
<400> 30
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
85 90 95
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 31
<211> 123
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H10
<400> 31
Gln Gln Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Glu Gly
1 5 10 15
Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Ile Ala Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Ala Ser Ser Thr Thr Val
65 70 75 80
Thr Leu Gln Met Thr Ser Leu Ala Ala Ala Asp Thr Ala Thr Tyr Phe
85 90 95
Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn Leu
100 105 110
Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 32
<211> 110
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> L7
<400> 32
Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile
35 40 45
Phe Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu Cys
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
85 90 95
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Glu Val Val Val
100 105 110
<210> 33
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H11
<400> 33
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 34
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H1L1
<400> 34
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser
180 185 190
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 35
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H1L2
<400> 35
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 36
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H1L3
<400> 36
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 37
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H1L4
<400> 37
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 38
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H1L5
<400> 38
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 39
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H3L2
<400> 39
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ala Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 40
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H3L3
<400> 40
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ala Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 41
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H3L4
<400> 41
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ala Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 42
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 42
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ala Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 43
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H4L1
<400> 43
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ser Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser
180 185 190
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 44
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H4L2
<400> 44
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ser Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 45
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H4L3
<400> 45
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ser Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 46
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H4L4
<400> 46
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ser Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 47
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H4L5
<400> 47
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ser Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 48
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H4L6
<400> 48
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ser Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr
180 185 190
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 49
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H5L1
<400> 49
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser
180 185 190
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 50
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H5L2
<400> 50
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 51
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H5L3
<400> 51
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 52
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H5L4
<400> 52
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 53
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H5L5
<400> 53
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 54
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H5L6
<400> 54
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr
180 185 190
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 55
<211> 249
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H6L1
<400> 55
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Tyr Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser
180 185 190
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile
245
<210> 56
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H6L2
<400> 56
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Tyr Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 57
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H6L3
<400> 57
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Tyr Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 58
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H6L4
<400> 58
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Tyr Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 59
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H6L5
<400> 59
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Tyr Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 60
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H6L6
<400> 60
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Tyr Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr
180 185 190
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 61
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H7L1
<400> 61
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
His Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser
180 185 190
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 62
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H7L2
<400> 62
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
His Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 63
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H7L3
<400> 63
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
His Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 64
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H7L4
<400> 64
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
His Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 65
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H7L5
<400> 65
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
His Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 66
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H8L2
<400> 66
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 67
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H8L3
<400> 67
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 68
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H8L4
<400> 68
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 69
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H8L5
<400> 69
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 70
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H8L6
<400> 70
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr
180 185 190
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 71
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H9L5
<400> 71
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr
180 185 190
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 72
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H9L6
<400> 72
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr
180 185 190
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 73
<211> 250
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H10L8
<400> 73
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn
100 105 110
Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr
180 185 190
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr
210 215 220
Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
245 250
<210> 74
<211> 248
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> H9L7
<400> 74
Gln Gln Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Glu Gly
1 5 10 15
Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Asp Leu Gly Phe Tyr
20 25 30
Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Ile Ala Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser
50 55 60
Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Ala Ser Ser Thr Thr Val
65 70 75 80
Thr Leu Gln Met Thr Ser Leu Ala Ala Ala Asp Thr Ala Thr Tyr Phe
85 90 95
Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn Leu
100 105 110
Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln
130 135 140
Thr Pro Ala Ser Val Ser Glu Pro Val Gly Gly Thr Val Thr Ile Lys
145 150 155 160
Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln Gln
165 170 175
Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile Phe Gly Ala Ser Thr Leu
180 185 190
Ala Ser Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu
195 200 205
Tyr Thr Leu Thr Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr
210 215 220
Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala Phe
225 230 235 240
Gly Gly Gly Thr Glu Val Val Val
245
<210> 75
<211> 21
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> CD8 leader amino acid sequence
<400> 75
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 76
<211> 45
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> CD8 hinge region amino acid sequence
<400> 76
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 77
<211> 24
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> CD8 transmembrane amino acid sequence
<400> 77
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 78
<211> 42
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> 4-1BB intracellular domain amino acid sequence
<400> 78
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 79
<211> 112
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> CD3 Domain amino acid sequence
<400> 79
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 80
<211> 494
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> H5L3-CAR
<400> 80
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Asp Leu Gly Phe Tyr Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser
65 70 75 80
Thr Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp
85 90 95
Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser
115 120 125
Thr Tyr Tyr Leu Asn Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
165 170 175
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
180 185 190
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
195 200 205
Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
225 230 235 240
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
245 250 255
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr
260 265 270
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
275 280 285
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
290 295 300
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
305 310 315 320
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
325 330 335
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
340 345 350
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
355 360 365
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
370 375 380
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
385 390 395 400
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
405 410 415
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
420 425 430
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
435 440 445
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
450 455 460
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
465 470 475 480
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 81
<211> 494
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> H5L4-CAR
<400> 81
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Asp Leu Gly Phe Tyr Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser
65 70 75 80
Thr Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp
85 90 95
Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser
115 120 125
Thr Tyr Tyr Leu Asn Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
165 170 175
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
180 185 190
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
195 200 205
Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
225 230 235 240
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
245 250 255
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr
260 265 270
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
275 280 285
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
290 295 300
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
305 310 315 320
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
325 330 335
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
340 345 350
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
355 360 365
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
370 375 380
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
385 390 395 400
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
405 410 415
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
420 425 430
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
435 440 445
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
450 455 460
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
465 470 475 480
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 82
<211> 494
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> H5L5-CAR
<400> 82
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Asp Leu Gly Phe Tyr Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser
65 70 75 80
Thr Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp
85 90 95
Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser
115 120 125
Thr Tyr Tyr Leu Asn Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
165 170 175
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser Tyr
180 185 190
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
195 200 205
Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
225 230 235 240
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
245 250 255
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr
260 265 270
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
275 280 285
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
290 295 300
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
305 310 315 320
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
325 330 335
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
340 345 350
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
355 360 365
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
370 375 380
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
385 390 395 400
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
405 410 415
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
420 425 430
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
435 440 445
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
450 455 460
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
465 470 475 480
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 83
<211> 494
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> H6L4-CAR
<400> 83
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Asp Leu Gly Phe Tyr Tyr Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser
65 70 75 80
Thr Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp
85 90 95
Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser
115 120 125
Thr Tyr Tyr Leu Asn Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
165 170 175
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
180 185 190
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
195 200 205
Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
225 230 235 240
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
245 250 255
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr
260 265 270
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
275 280 285
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
290 295 300
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
305 310 315 320
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
325 330 335
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
340 345 350
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
355 360 365
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
370 375 380
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
385 390 395 400
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
405 410 415
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
420 425 430
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
435 440 445
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
450 455 460
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
465 470 475 480
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 84
<211> 494
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> H6L5-CAR
<400> 84
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Asp Leu Gly Phe Tyr Tyr Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser
65 70 75 80
Thr Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp
85 90 95
Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser
115 120 125
Thr Tyr Tyr Leu Asn Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
165 170 175
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser Tyr
180 185 190
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
195 200 205
Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
225 230 235 240
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
245 250 255
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr
260 265 270
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
275 280 285
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
290 295 300
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
305 310 315 320
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
325 330 335
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
340 345 350
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
355 360 365
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
370 375 380
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
385 390 395 400
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
405 410 415
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
420 425 430
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
435 440 445
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
450 455 460
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
465 470 475 480
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 85
<211> 494
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> H6L6-CAR
<400> 85
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Asp Leu Gly Phe Tyr Tyr Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser
65 70 75 80
Thr Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp
85 90 95
Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser
115 120 125
Thr Tyr Tyr Leu Asn Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
165 170 175
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
180 185 190
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
195 200 205
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
225 230 235 240
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
245 250 255
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr
260 265 270
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
275 280 285
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
290 295 300
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
305 310 315 320
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
325 330 335
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
340 345 350
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
355 360 365
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
370 375 380
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
385 390 395 400
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
405 410 415
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
420 425 430
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
435 440 445
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
450 455 460
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
465 470 475 480
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 86
<211> 494
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> H11L8-CAR
<400> 86
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Asp Leu Gly Phe Tyr Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser
65 70 75 80
Thr Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp
85 90 95
Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser
115 120 125
Thr Tyr Tyr Leu Asn Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
165 170 175
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr
180 185 190
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
195 200 205
Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
225 230 235 240
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser
245 250 255
Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr
260 265 270
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
275 280 285
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
290 295 300
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
305 310 315 320
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
325 330 335
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
340 345 350
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
355 360 365
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
370 375 380
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
385 390 395 400
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
405 410 415
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
420 425 430
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
435 440 445
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
450 455 460
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
465 470 475 480
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 87
<211> 492
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> H10L7-CAR
<400> 87
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Gln Gln Leu Glu Glu Ser Gly Gly Gly Leu
20 25 30
Val Lys Pro Glu Gly Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe
35 40 45
Asp Leu Gly Phe Tyr Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Cys Ile Tyr Thr Ala Gly Ser Gly Ser
65 70 75 80
Thr Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Ala
85 90 95
Ser Ser Thr Thr Val Thr Leu Gln Met Thr Ser Leu Ala Ala Ala Asp
100 105 110
Thr Ala Thr Tyr Phe Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr
115 120 125
Tyr Tyr Leu Asn Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
145 150 155 160
Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly Gly
165 170 175
Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu
180 185 190
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile Phe
195 200 205
Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly Ser
210 215 220
Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu Cys Ala
225 230 235 240
Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn
245 250 255
Asn Trp His Ala Phe Gly Gly Gly Thr Glu Val Val Val Thr Thr Thr
260 265 270
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
275 280 285
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
290 295 300
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
305 310 315 320
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
325 330 335
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
340 345 350
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
355 360 365
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
370 375 380
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
385 390 395 400
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
405 410 415
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
420 425 430
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
435 440 445
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
450 455 460
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
465 470 475 480
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 88
<211> 1485
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> gene
<223> H5L5-CAR nucleic acid sequence
<400> 88
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggaggtgc agctgctcga aagcggcgga ggactggtgc aacccggcgg aagcctcaga 120
ctgagctgtg ctgccagcgg cttcgatctg ggcttctact tctacgcttg ttgggtgagg 180
caagcccccg gcaaaggact ggagtgggtg agctgcatct atactgccgg ctccggctcc 240
acatactatg ccagctgggc caagggaagg ttcactatct ctagggacaa cagcaagaac 300
actctgtatc tgcagatgaa ctctctgagg gctgaggata ctgccgtcta ttactgcgcc 360
agaagcactg ccaacacaag gagcacttac tatctgaata cttggggcca aggcactctg 420
gtcactgtga gcagcggcgg aggcggatca ggtggtggcg gatctggagg tggcggaagc 480
gatatccaga tgactcagtc cccttcctct ctgagcgcta gcgtgggaga cagagtgact 540
atcacatgca gggccagcca gaggatctcc agctatctga gctggtacca gcagaagccc 600
ggcaaggtgc ctaagctgct gatctatggc gcctccactc tggctagcgg agtgccttcc 660
agatttagcg gcagcggaag cggcactgac ttcactctga ctatcagctc tctgcagcca 720
gaggacgtgg ctacttacta ctgccagagc tacgcctact tcgacagcaa caactggcac 780
gcctttggcg gcggcactaa ggtcgagatc aaaaccacga cgccagcgcc gcgaccacca 840
acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900
gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 960
gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1020
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1080
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1140
gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1200
cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1260
cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1320
tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1380
gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1440
gacacctacg acgcccttca catgcaggcc ctgccccctc gctga 1485
<210> 89
<211> 1485
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> gene
<223> H6L6-CAR nucleic acid sequence
<400> 89
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggaggtgc agctgctcga aagcggcgga ggactggtgc aacccggcgg aagcctcaga 120
ctgagctgtg ctgccagcgg cttcgatctg ggcttctact actacgcttg ttgggtgagg 180
caagcccccg gcaaaggact ggagtgggtg agctgcatct atactgccgg ctccggctcc 240
acatactatg ccagctgggc caagggaagg ttcactatct ctagggacaa cagcaagaac 300
actctgtatc tgcagatgaa ctctctgagg gctgaggata ctgccgtcta ttactgcgcc 360
agaagcactg ccaacacaag gagcacttac tatctgaatc tgtggggcca aggcactctg 420
gtcactgtga gcagcggcgg aggcggatca ggtggtggcg gatctggagg tggcggaagc 480
gatatccaga tgactcagtc cccttcctct ctgagcgcta gcgtgggaga cagagtgact 540
atcacatgcc aagccagcca gaggatctcc agctatctga gctggtacca gcagaagccc 600
ggcaaggtgc ctaagctgct gatctatgct gcctccactc tgcagagcgg agtgccttcc 660
agatttagcg gcagcggaag cggcactgac ttcactctga ctatcagctc tctgcagcca 720
gaggacgtgg ctacttacta ctgccagagc tacgcctact tcgacagcaa caactggcac 780
gcctttggcg gcggcactaa ggtcgagatc aaaaccacga cgccagcgcc gcgaccacca 840
acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900
gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 960
gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1020
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1080
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1140
gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1200
cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1260
cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1320
tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1380
gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1440
gacacctacg acgcccttca catgcaggcc ctgccccctc gctga 1485
<210> 90
<211> 549
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> promoter
<223> EF1 alpha promoter
<400> 90
ctagtggatc tgcgatcgct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt 60
ccccgagaag ttggggggag gggtcggcaa ttgaacgggt gcctagagaa ggtggcgcgg 120
ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga 180
accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag 240
aacacagctg aagcttcgag gggctcgcat ctctccttca cgcgcccgcc gccctacctg 300
aggccgccat ccacgccggt tgagtcgcgt tctgccgcct cccgcctgtg gtgcctcctg 360
aactgcgtcc gccgtctagg taagtttaaa gctcaggtcg agaccgggcc tttgtccggc 420
gctcccttgg agcctaccta gactcagccg gctctccacg ctttgcctga ccctgcttgc 480
tcaactctac gtctttgttt cgttttctgt tctgcgccgt tacagatcca agctgtgacc 540
ggcgcctac 549
<210> 91
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> HCDR1 general formula
<220>
<221> DOMAIN
<222> (3)..(3)
<223> Xaa is selected from Phe, tyr or His.
<220>
<221> UNSURE
<222> (3)..(3)
<223> The 'Xaa' at location 3 stands for Gln, Arg, Pro, or Leu.
<400> 91
Phe Tyr Xaa Tyr Ala Cys
1 5
<210> 92
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> HCDR2 general formula
<220>
<221> DOMAIN
<222> (14)..(14)
<223> Xaa is selected from Ser or ASP.
<220>
<221> DOMAIN
<222> (15)..(15)
<223> Xaa is selected from Trp or ser.
<220>
<221> DOMAIN
<222> (18)..(18)
<223> Xaa is selected from Ala or Val.
<220>
<221> UNSURE
<222> (14)..(14)
<223> The 'Xaa' at location 14 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (15)..(15)
<223> The 'Xaa' at location 15 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (16)..(16)
<223> The 'Xaa' at location 16 stands for Gln, Arg, Pro, or Leu.
<400> 92
Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Xaa Xaa Xaa
1 5 10 15
Lys Gly
<210> 93
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> HCDR3 general formula
<220>
<221> DOMAIN
<222> (3)..(3)
<223> Xaa is selected from Ser or Ala.
<220>
<221> DOMAIN
<222> (14)..(14)
<223> Xaa is selected from Thr or Leu.
<220>
<221> UNSURE
<222> (3)..(3)
<223> The 'Xaa' at location 3 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (13)..(13)
<223> The 'Xaa' at location 13 stands for Gln, Arg, Pro, or Leu.
<400> 93
Ser Thr Xaa Asn Thr Arg Ser Thr Tyr Tyr Leu Asn Xaa
1 5 10
<210> 94
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> LCDR1 formula
<220>
<221> DOMAIN
<222> (1)..(1)
<223> Xaa is selected from Gln or Arg.
<220>
<221> UNSURE
<222> (1)..(1)
<223> The 'Xaa' at location 1 stands for Gln, Arg, Pro, or Leu.
<400> 94
Xaa Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser
1 5 10
<210> 95
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> LCDR2 general formula
<220>
<221> DOMAIN
<222> (4)..(4)
<223> Xaa is selected from Ser or Thr.
<220>
<221> DOMAIN
<222> (6)..(6)
<223> Xaa is selected from Ala or Gln.
<220>
<221> UNSURE
<222> (1)..(1)
<223> The 'Xaa' at location 1 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (4)..(4)
<223> The 'Xaa' at location 4 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (6)..(6)
<223> The 'Xaa' at location 6 stands for Gln, Arg, Pro, or Leu.
<400> 95
Xaa Ala Ser Xaa Leu Xaa Ser
1 5
<210> 96
<211> 488
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> N2 (CD 19 CAR) amino acid sequence
<400> 96
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
115 120 125
Arg Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser
145 150 155 160
Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp
165 170 175
Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp
180 185 190
Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu
195 200 205
Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe
210 215 220
Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys
225 230 235 240
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly
245 250 255
Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg
260 265 270
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
275 280 285
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
290 295 300
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
305 310 315 320
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg
325 330 335
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
340 345 350
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
355 360 365
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
370 375 380
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
385 390 395 400
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
405 410 415
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg
485
<210> 97
<211> 341
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> MSLN-R3-hFc4
<400> 97
Met Asn Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly
1 5 10 15
Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met
20 25 30
Asp Leu Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu
35 40 45
Thr Val Ala Glu Val Gln Lys Leu Leu Gly Pro His Val Glu Gly Leu
50 55 60
Lys Ala Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gln
65 70 75 80
Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile
85 90 95
Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser
100 105 110
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
115 120 125
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
130 135 140
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
145 150 155 160
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
165 170 175
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
180 185 190
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
195 200 205
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
210 215 220
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
225 230 235 240
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
245 250 255
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
260 265 270
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
275 280 285
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
290 295 300
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
305 310 315 320
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
325 330 335
Leu Ser Leu Gly Lys
340
<210> 98
<211> 451
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> PEPTIDE
<223> RS7 antibody heavy chain
<400> 98
Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly
100 105 110
Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210> 99
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<221> DOMAIN
<223> RS7 antibody light chain
<400> 99
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210

Claims (34)

1. A Chimeric Antigen Receptor (CAR) molecule comprising:
i) An anti-mesothelin binding domain,
ii) a transmembrane domain, and
iii) An intracellular signaling domain;
wherein the anti-mesothelin binding domain is an scFv comprising a light chain variable region and a heavy chain variable region, wherein:
a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3.
2. The CAR molecule of claim 1, wherein the scFv comprises:
as set forth in SEQ ID NO:30 and the light chain variable region as set forth in SEQ ID NO: 21.
3. The CAR molecule of claim 1, wherein the anti-mesothelin binding domain comprises the amino acid sequence set forth in SEQ ID NO: 60.
4. The CAR molecule of claim 1, wherein the transmembrane domain comprises a transmembrane domain of any one of the proteins selected from the group consisting of: the α, β or ζ chain of T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
5. The CAR molecule of claim 4, wherein the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 77.
6. The CAR molecule of claim 1, wherein the anti-mesothelin binding domain is linked to the transmembrane domain by a hinge region.
7. The CAR molecule of claim 6, wherein the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 76.
8. The CAR molecule of claim 1, wherein the intracellular signaling domain is a functional signaling domain of cd3ζ.
9. The CAR molecule of claim 8, wherein the functional signaling domain of cd3ζ comprises the amino acid sequence of SEQ ID NO: 79.
10. The CAR molecule of claim 1, wherein the intracellular signaling domain further comprises a co-stimulatory domain.
11. The CAR molecule of claim 10, wherein the co-stimulatory domain comprises a functional signaling domain selected from any one of the following proteins: OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1, CD278 and 4-1BB.
12. The CAR molecule of claim 11, wherein the co-stimulatory domain is a functional signaling domain of 4-1BB.
13. The CAR molecule of claim 12, wherein the functional signaling domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO: 78.
14. The CAR molecule of claim 1, wherein the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:78 and the sequence shown as SEQ ID NO: 79.
15. The CAR molecule of claim 1, further comprising a leader sequence.
16. The CAR molecule of claim 15, wherein the leader sequence comprises the sequence set forth in SEQ ID NO: 75.
17. The CAR molecule of claim 1, comprising the amino acid sequence set forth in SEQ ID NO: 85.
18. An antibody that specifically binds mesothelin comprising a light chain variable region and a heavy chain variable region, wherein:
a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:15 and SEQ ID NO:6 LCDR1, LCDR2 and LCDR3, and
A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3.
19. The antibody that specifically binds to mesothelin according to claim 18, comprising a light chain variable region and a heavy chain variable region, wherein:
the sequence of the light chain variable region is shown in SEQ ID NO:30 and the sequence of the heavy chain variable region shown and described as SEQ ID NO: 21.
20. The antibody that specifically binds to mesothelin according to claim 19, which is a single chain antibody, wherein said single chain antibody comprises:
as set forth in SEQ ID NO:21, and a heavy chain variable region shown in FIG. 21, and
as set forth in SEQ ID NO:30, and a light chain variable region shown in seq id no.
21. The antibody that specifically binds to mesothelin according to claim 18, comprising the amino acid sequence as set forth in SEQ ID NO: 60.
22. A nucleic acid molecule encoding the CAR molecule of any one of claims 1 to 17 or the antibody of any one of claims 18 to 21 that specifically binds mesothelin.
23. The nucleic acid molecule of claim 22, comprising the sequence set forth in SEQ ID NO: 89.
24. A recombinant vector comprising the nucleic acid molecule of claim 22 or 23.
25. The recombinant vector of claim 24, wherein said recombinant vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, and retroviral vector.
26. The recombinant vector of claim 25, further comprising a promoter.
27. The recombinant vector of claim 26, wherein said promoter is EF-1 promoter.
28. The recombinant vector of claim 27, wherein the EF-1 promoter comprises the amino acid sequence as set forth in SEQ ID NO: 90.
29. A cell comprising the recombinant vector of any one of claims 24 to 28.
30. The cell of claim 29, wherein the cell is an immune effector cell and expresses the CAR molecule of any one of claims 1 to 17.
31. The cell of claim 30, wherein the immune effector cell is an NK cell, a macrophage, a dendritic cell, or a T cell.
32. The cell of claim 31, wherein the immune effector cell is a cd8+ T cell or a cd4+ T cell.
33. A pharmaceutical composition comprising a therapeutically effective amount of an antibody of any one of claims 18 to 21 that specifically binds mesothelin, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
34. Use of a cell expressing a CAR according to any one of claims 1 to 17, or an antibody that specifically binds mesothelin according to any one of claims 18 to 21, or a pharmaceutical composition according to claim 33, in the manufacture of a medicament for the treatment or prophylaxis of cancer, wherein the cancer is selected from: mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, cholangiocarcinoma, pancreatic cancer, pancreatic ductal adenoma, ovarian cancer, colorectal cancer, breast cancer, and bladder cancer.
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CN101951946A (en) * 2007-10-01 2011-01-19 百时美施贵宝公司 Human antibodies that bind mesothelin, and uses thereof
CN109306014A (en) * 2017-07-27 2019-02-05 上海细胞治疗研究院 A kind of Chimeric antigen receptor modification T cell and application thereof targeting mesothelin
CN110055269A (en) * 2019-03-20 2019-07-26 英威福赛生物技术有限公司 People's mesothelin Chimeric antigen receptor, its T cell and its preparation method and application

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Publication number Priority date Publication date Assignee Title
CN101951946A (en) * 2007-10-01 2011-01-19 百时美施贵宝公司 Human antibodies that bind mesothelin, and uses thereof
CN109306014A (en) * 2017-07-27 2019-02-05 上海细胞治疗研究院 A kind of Chimeric antigen receptor modification T cell and application thereof targeting mesothelin
CN110055269A (en) * 2019-03-20 2019-07-26 英威福赛生物技术有限公司 People's mesothelin Chimeric antigen receptor, its T cell and its preparation method and application

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