CN113698492A

CN113698492A - Human mesothelin chimeric antigen receptor and uses thereof

Info

Publication number: CN113698492A
Application number: CN202110557515.1A
Authority: CN
Inventors: 胡齐悦; 童建松; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2020-05-22
Filing date: 2021-05-21
Publication date: 2021-11-26
Anticipated expiration: 2041-05-21
Also published as: CN113698492B

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 uses thereof

Technical Field

The present disclosure relates to antibodies that specifically bind mesothelin, T cells expressing a Chimeric Antigen Receptor (CAR) that specifically binds mesothelin, and their use 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 Receptors (CARs) are molecules comprising three basic units: (1) an extracellular antigen binding motif, (2) a ligation/transmembrane motif, and (3) an intracellular T cell signaling motif, which is specific for a selected antigen. Targeted T cell therapies using CARs have achieved clinical success in treating some hematologic malignancies, however, the use of CAR-expressing T cell therapies for the treatment of solid tumors remains difficult.

Human Mesothelin (MSLN) is a 40kDa cell surface Glycosylphosphatidylinositol (GPI) -linked glycoprotein. This protein is synthesized as a 70kD precursor and then proteolytically processed. The 30KD amino-terminus of mesothelin is secreted and is called Megakaryocyte Potentiator (MPF) (Yamaguchi et al, J.biol.chem.269: 805808, 1994). The 40kD carboxy terminus remains bound to the membrane as mature mesothelin (Chang et al, Natl.Acad.Sci.USA 93: 136140, 1996).

MSLNs are not essential in normal tissues, and are rarely found in normal adult tissues including mesothelium, but are abnormally abundantly expressed in, for example, mesothelioma, pancreatic cancer and ovarian cancer (Chang K, Pastan I.molecular cloning of mesothelin, a differentiation antisense expression on mesothelium, mesotheliomas, and ovarian cancer. Proc Natl Acad Sci USA 1996; 93: 136-40.). Excessive mesothelin expression is first found in mesothelioma and ovarian Cancer, and then subsequently in lung, esophageal, pancreatic, gastric, biliary, endometrial, thymus, colon, breast cancers (Morello A, Sadelain M, Adusumii P S. Mesothelin-Targeted CARs: drying T cells to Solid Tumors [ J ]. Cancer Discovery,2015,6 (2)). 90% of epithelioid malignant pleural mesothelioma, 69% lung adenocarcinoma, 60% breast Cancer, 46% esophageal Cancer express mesothelin (Morello A, Sadelain M, Adusumili PS. Mesothelin-Targeted CARs: Driving T cells to Solid Tumors [ J ]. Cancer Discovery,2015,6 (2)). Mesothelin is also expressed in the more aggressive lung, breast (triple negative breast) and esophageal (high grade dysplasia and adenocarcinoma) cancers. Therefore, mesothelin, as a tumor antigen, is a good target for CAR-T.

Therefore, the continuous research and development of CAR-T aiming at MSLN has important significance for improving the treatment effect of tumors. Currently, CART for MSLN is disclosed in the following patents, such as WO2013063419, CN109097396, WO2017112741, WO2020043152, and the like.

Disclosure of Invention

The present disclosure provides a chimeric antigen receptor against MSLN (MSLN-CAR) with a novel sequence that was experimentally verified to bind specifically to human MSLN and effectively kill tumor target cells in vivo and in vitro.

In some embodiments, the Chimeric Antigen Receptor (CAR) molecule comprises:

i) (ii) 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 such as X₁ASQRISSYLS (SEQ ID NO: 94);

LCDR2 such as X₂ASX₃LX₄S (SEQ ID NO: 95);

LCDR3 is shown as QSYAYFDSNNWHA (SEQ ID NO: 6); and

the heavy chain variable region comprises an HCDR as shown below:

HCDR1 such as FYX₅YAC (SEQ ID NO: 91);

HCDR2 such as CIYTAGSGSTYYAX₆X₇X₈KG(SEQ ID NO: 92) shown;

HCDR3 such as STX₉NTRSTYYLNX₁₀(SEQ ID NO: 93);

wherein, X₁Selected from Q or R, X₂Selected from A or G, X₃Selected from S or T, X₄Selected from A or Q, X₅Selected from F, Y or H, X₆Selected from S or D, X₇Selected from W or S, X₈Selected from A or V, X₉Selected from S or A, X₁₀Is selected from T or L.

In some embodiments, the CAR molecule as described above, whose 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 sequences shown as SEQ ID NO: 4. SEQ ID NO: 14 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 3;

b) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

c) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

d) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 14 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 11;

e) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

f) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

g) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

h) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 14 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 12;

i) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

j) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

k) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

l) the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO: 14 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 7. SEQ ID NO: 2 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 3;

m) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

n) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

o) the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

p) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 14 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 8. SEQ ID NO: 2 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 3;

q) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

r) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

s) the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 1. SEQ ID NO: 9 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 3;

t) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

u) the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

v) the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 1. SEQ ID NO: 10 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 3; or

w) the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 1. SEQ ID NO: 10 and SEQ ID NO: 3 HCDR1, HCDR2 and HCDR 3.

the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 12; or

The light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 7. SEQ ID NO: 2 and SEQ ID NO: 3 HCDR1, HCDR2 and HCDR 3.

In some embodiments, the CAR as described above, whose anti-mesothelin binding domain comprises a light chain variable region and a heavy chain variable region, wherein:

the light chain variable region sequence is shown as SEQ ID NO: 25. 26, 27, 28, 29, 30 or 32; and/or

The heavy chain variable region sequence is shown as SEQ ID NO: 16. 17, 18, 19, 20, 21, 22, 23, 24, 31 or 33.

a1) the light chain variable region sequence is shown as SEQ ID NO: 25 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and

the heavy chain variable region sequence is shown as SEQ ID NO: 16. 19, 20, 21 or 22, or a variant of SEQ ID NO: 16. 19, 20, 21 or 22 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, respectively;

b1) the light chain variable region sequence is shown as SEQ ID NO: 26, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and

the heavy chain variable region sequence is shown as SEQ ID NO: 16. 18, 19, 20, 21, 22, 23 or 33, or a variant of SEQ ID NO: 16. 18, 19, 20, 21, 22, 23 or 33 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, respectively;

c1) the light chain variable region sequence is shown as SEQ ID NO: 27, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and

the heavy chain variable region sequence is shown as SEQ ID NO: 16. 18, 19, 20, 21, 22 or 23, or a variant of SEQ ID NO: 16. 18, 19, 20, 21, 22 or 23 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, respectively;

d1) the light chain variable region sequence is shown as SEQ ID NO: 28, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and

e1) the light chain variable region sequence is shown as SEQ ID NO: 29, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and

the heavy chain variable region sequence is shown as SEQ ID NO: 16. 18, 19, 20, 21, 22, 23 or 24, or a variant of SEQ ID NO: 16. 18, 19, 20, 21, 22, 23 or 24 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, respectively;

f1) the light chain variable region sequence is shown as SEQ ID NO: 30, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and

the heavy chain variable region sequence is shown as SEQ ID NO: 19. 20, 21, 23 or 24, or a variant of SEQ ID NO: 19. 20, 21, 23 or 24, respectively, have at least 95% sequence identity; or

g1) The light chain variable region sequence is shown as SEQ ID NO: 32, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and

the heavy chain variable region sequence is shown as SEQ ID NO: 31, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

In some embodiments, the CAR as described above, whose anti-mesothelin binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 29 and the variable region of the light chain as set forth in SEQ ID NO: 20, a heavy chain variable region; or

Comprises the amino acid sequence shown as SEQ ID NO: 30 and the variable region of the light chain as shown in SEQ ID NO: 21, or a heavy chain variable region as shown in figure 21.

In some embodiments, the CAR as described above, whose anti-mesothelin binding domain is an scFv.

In some embodiments, the CAR as previously described, wherein the anti-mesothelin binding domain scFv comprises:

as shown in SEQ ID NO: 25. 26, 27, 28, 29, 30, or 32, and/or

As shown in SEQ ID NO: 16. 17, 18, 19, 20, 21, 22, 23, 24, 31, or 33.

as shown in SEQ ID NO: 29 and the variable region of the light chain as set forth in SEQ ID NO: 20, a heavy chain variable region; or

As shown in SEQ ID NO: 30 and the variable region of the light chain as shown in SEQ ID NO: 21, or a heavy chain variable region as shown in figure 21.

In some embodiments, the CAR as described above, wherein said anti-mesothelin binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 34-74.

In some embodiments, the CAR as described above, wherein said anti-mesothelin binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 51. 52, 53, 58, 59 or 60.

In some embodiments, the CAR as described above, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from any one of: the α, β or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154.

In some embodiments, the CAR as described above, wherein the transmembrane domain comprises SEQ ID NO: 77.

In some embodiments, the CAR as described above, wherein the anti-mesothelin binding domain is connected to the transmembrane domain by a hinge region.

In some embodiments, the CAR as described above, wherein the hinge region comprises SEQ ID NO: 76, respectively, and a sequence shown in fig. 76.

In some embodiments, the CAR as described above, wherein said intracellular signaling domain is a functional signaling domain of CD3 ζ;

in some embodiments, the CAR as described above, wherein said functional signaling domain of CD3 ζ comprises a sequence as set forth in SEQ ID NO: 79, or a pharmaceutically acceptable salt thereof.

In some embodiments, the CAR as described above, wherein the intracellular signaling domain further comprises a costimulatory domain.

In some embodiments, the CAR as described above, wherein the co-stimulatory domain comprises a functional signaling domain of a protein selected from any one of: OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), CD278 (also known as "ICOS"), and 4-1BB (CD 137).

In some embodiments, the CAR as described above, wherein the co-stimulatory domain is a functional signaling domain of 4-1 BB.

In some embodiments, the CAR as described above, wherein said functional signaling domain of 4-1BB comprises an amino acid sequence as set forth in SEQ ID NO: 78, or a sequence shown in seq id no.

In some embodiments, the CAR as described above, wherein the intracellular signaling domain comprises an amino acid sequence as set forth in SEQ id no: 78 and/or the sequence as shown in SEQ ID NO: 79, or a pharmaceutically acceptable salt thereof.

In some embodiments, the CAR as described above, further comprising a leader sequence.

In some embodiments, the CAR as described above, wherein the leader sequence comprises the sequence set forth as SEQ ID NO: 75, or a sequence shown in seq id no.

In some embodiments, the CAR as previously described, comprising a sequence as set forth in SEQ ID NO: 80-87, or a fragment thereof.

In some embodiments, the CAR as previously described, comprising a sequence as set forth in SEQ ID NO: 82 or 85, respectively.

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.

In some embodiments, the antibody that specifically binds mesothelin, which 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 such as X₁ASQRISSYLS (SEQ ID NO: 94);

LCDR2 such as X₂ASX₃LX₄S (SEQ ID NO: 95);

LCDR3 is shown as QSYAYFDSNNWHA (SEQ ID NO: 6); and

the heavy chain variable region comprises an HCDR as shown below:

HCDR1 such as FYX₅YAC (SEQ ID NO: 91);

HCDR2 such as CIYTAGSGSTYYAX₆X₇X₈KG (SEQ ID NO: 92);

HCDR3 such as STX₉NTRSTYYLNX₁₀(SEQ ID NO: 93);

In some embodiments, the antibody that specifically binds mesothelin as described above, comprising the light chain variable region and the heavy chain variable region of any one of:

c) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 14 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

d) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

e) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

f) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

g) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 14 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

h) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

i) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

j) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

k) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 14 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

l) the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

m) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

n) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

o) the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO: 14 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

p) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

q) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

r) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

s) the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

t) the light chain variable region comprises the amino acid sequences shown as SEQ ID NO: 4. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

u) the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

v) the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

In some embodiments, the antibody that specifically binds mesothelin as described above, wherein said antibody that specifically binds mesothelin comprises:

a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 13. SEQ ID NO: 5 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

A light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 4. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in FIG. 6, and

In some embodiments, an antibody that specifically binds mesothelin as described above, comprising a light chain variable region and a heavy chain variable region, wherein:

the light chain variable region sequence is shown as SEQ ID NO: 25. 26, 27, 28, 29 or 30; and/or

The heavy chain variable region sequence is shown as SEQ ID NO: 16. 17, 18, 19, 20, 21, 22, 23 or 24.

a1) the light chain variable region sequence is shown as SEQ ID NO: 25, or has at least 95% sequence identity thereto; and

the heavy chain variable region sequence is shown as SEQ ID NO: 16. 19, 20, 21 or 22, or a variant of 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, having at least 95% sequence identity thereto; and

the heavy chain variable region sequence is shown as SEQ ID NO: 16. 18, 19, 20, 21, 22 or 23, or a variant of SEQ ID NO: 16. 18, 19, 20, 21, 22 or 23 have 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 has at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and

d1) the light chain variable region sequence is shown as SEQ ID NO: 28, or has at least 95% sequence identity thereto; and

e1) the light chain variable region sequence is shown as SEQ ID NO: 29, or has at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; and

the heavy chain variable region sequence is shown as SEQ ID NO: 16. 18, 19, 20, 21, 22, 23 or 24, or a variant of SEQ ID NO: 16. 18, 19, 20, 21, 22, 23 or 24 have at least 95%, 96%, 97%, 98% or 99% sequence identity; or

f1) The light chain variable region sequence is shown as SEQ ID NO: 30, or has at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; and

the heavy chain variable region sequence is shown as SEQ ID NO: 19. 20, 21, 23 or 24, or a variant of SEQ ID NO: 19. 20, 21, 23 or 24 have at least 95%, 96%, 97%, 98% or 99% sequence identity.

the light chain variable region sequence is shown as SEQ ID NO: 29 and the heavy chain variable region sequence is as shown in SEQ ID NO: 20 is shown in the figure; or

The light chain variable region sequence is shown as SEQ ID NO: 30 and the heavy chain variable region sequence is as shown in SEQ ID NO: shown at 21.

In some embodiments, the antibody that specifically binds mesothelin as described above is a single chain antibody.

In some embodiments, a single chain antibody that specifically binds mesothelin as described above comprises:

as shown in SEQ ID NO: 16. 17, 18, 19, 20, 21, 22, 23, or 24, and/or

As shown in SEQ ID NO: 25. 26, 27, 28, 29 or 30.

In some embodiments, an antibody that specifically binds mesothelin as described above, 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 above, comprising an amino acid sequence as set forth in SEQ ID NO: 51. 52, 53, 58, 59 or 60.

The disclosure also provides a nucleic acid molecule encoding any one of the CARs of the preceding claims or an antibody that specifically binds mesothelin of any of the preceding claims.

In some embodiments, wherein the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 88 or 89.

The 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 a DNA, an RNA, a plasmid, a lentiviral vector, an adenoviral vector, and a retroviral vector.

In some embodiments, wherein the recombinant vector further comprises a promoter.

In some embodiments, the promoter is the EF-1 promoter.

In some embodiments, the EF-1 promoter comprises the amino acid sequence of SEQ ID NO: 90, or a sequence shown in seq id no.

The 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 a CAR as described in any of the preceding.

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 to an immune effector cell.

The present disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of an antibody that specifically binds mesothelin of any one of the preceding, or a nucleic acid molecule of the preceding, or a cell of the preceding, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

The disclosure also provides use of a cell expressing a CAR as described in any preceding claim, or an antibody that specifically binds mesothelin as described above, or a nucleic acid molecule as described above, or a pharmaceutical composition as described above, in the manufacture of a medicament for the treatment or prevention of cancer; preferably, wherein said cancer is selected from: mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, cholangiocarcinoma, breast cancer, malignant pleural mesothelioma, non-small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, ductal adenoma of the pancreas, ovarian cancer, colorectal cancer, and bladder cancer; more preferably, wherein said cancer is associated with mesothelin expression.

In some embodiments, the disclosure provides a method of treating cancer comprising administering to a subject a therapeutically effective amount of a cell expressing an isolated CAR as described in any of the preceding, or an antibody that specifically binds mesothelin as described above, or a nucleic acid molecule as described above, or a pharmaceutical composition as described above; preferably, wherein said cancer is selected from: mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, cholangiocarcinoma, breast cancer, malignant pleural mesothelioma, non-small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, ductal adenoma of the pancreas, ovarian cancer, colorectal cancer, and bladder cancer; more preferably, wherein said cancer is associated with mesothelin expression.

In some embodiments, the disclosure provides a cell expressing an isolated CAR as described in any of the preceding, or an antibody that specifically binds mesothelin as described above, or a nucleic acid molecule as described above, or a pharmaceutical composition as described above, for use in the treatment of cancer; preferably, wherein said cancer is selected from: mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, cholangiocarcinoma, breast cancer, malignant pleural mesothelioma, non-small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, ductal adenoma of the pancreas, ovarian cancer, colorectal cancer, and bladder cancer; more preferably, wherein said cancer is associated with mesothelin expression.

Description of the drawings:

FIG. 1: different MSLN-scFv antibody cell levels bound to human MSLN assay results.

FIG. 2: results of MSLN-CAR mediated T cell activation experiments.

FIG. 3: FIG. 3A shows a MSLN-CART cell survival-time curve; FIG. 3B shows a MSLN-CART cell proliferation fold-time curve; FIG. 3C shows CAR positivity rate in MSLN-CART cells; FIG. 3D shows MFI of MSLN-CART cells.

FIG. 4: FIG. 4A shows MSLN-CART cell proliferation curves without antigen repeat stimulation; FIG. 4B shows the measured proliferation curve of MSLN-CART cells after repeated antigen 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 IFN γ during killing of target cells by MSLN-CART cells.

FIG. 7: FIG. 7A shows the change in body weight of mice; figure 7B shows the change in tumor volume in mice.

FIG. 8: persistent proliferation of MSLN-CART cells in mice.

Detailed Description

Definition of

In order that the disclosure may be more readily understood, 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 signal production. 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 and/or co-stimulatory molecules as defined below. In certain aspects, the sets of polypeptides are contiguous to each other. In certain embodiments, the set of polypeptides includes a dimerization switch that can couple the polypeptides to each other in the presence of a dimerization molecule, e.g., an antigen binding domain can be coupled to an intracellular signaling domain. In one aspect, the stimulatory molecule is a zeta chain that binds to a 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 co-stimulatory molecule is selected from the co-stimulatory molecules described herein, such as 4-1BB (i.e., CD137), CD27, and/or CD 28. 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 the cell membrane.

The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell to modulate the activity of the cell via a defined signaling pathway, either by generating second messengers or by acting as effectors in response to such messengers.

The term "mesothelin" refers to the 40kDa protein mesothelin, which is anchored to the cell membrane by a Glycosylphosphatidylinositol (GPI) linkage and an amino-terminal 31kDa abscission fragment (known as megakaryocyte enhancer factor, MPF). Both fragments contain N-glycosylation sites. The term also refers to soluble splice variants of the 40kDa carboxy-terminal fragment, also known as "soluble mesothelin/MPF-related". Preferably, the term refers to human mesothelin and naturally-cleaved portions thereof, e.g., expressed on cell membranes (e.g., cancer cell membranes), of GenBank accession No. AAH 03512.1.

An "antibody" as described in the present disclosure refers to an immunoglobulin, a complete full-length antibody being a tetrapeptide chain structure formed by two heavy chains and two light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in amino acid composition and arrangement, as well as antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. 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 the heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3.

Antibodies of the disclosure include human antibodies.

The term "murine antibody" is in this disclosure a monoclonal antibody against human MSLN prepared according to the knowledge and skill in the art. Preparation is performed by injecting a test subject with the MSLN antigen and then isolating the hybridoma expressing the antibody with the desired sequence or functional properties. In a preferred embodiment of the present disclosure, the murine anti-MSLN antibody may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or variant thereof.

The term "chimeric antibody" refers to an antibody obtained by fusing the variable region of an antibody of one species (e.g., mouse) to the constant region of an antibody of another species (e.g., human), and can reduce an immune response induced by a heterologous antibody. Illustratively, the chimeric antibody is established by establishing a hybridoma secreting a mouse-derived specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to needs, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of said MSLN chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof. The antibody heavy chain of the MSLN chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof, preferably comprises 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", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting CDR sequences of an antibody of a certain species (e.g., murine) into the framework of the variable region of a human antibody, i.e., the framework sequences of different types of human germline antibodies. Can overcome the heterogenous reaction induced by the chimeric antibody carrying a large amount of heterogenous protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA Sequences of genes such as the human heavy and light chain variable regions can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/VBase), as well as in Kabat, E.A. et al, 1991Sequences of Proteins of Immunological Interest, 5 th edition. To avoid reduced immunogenicity and reduced activity, the human antibody variable region framework sequences may be minimally back-mutated or back-mutated to retain activity. Humanized antibodies of the disclosure also include humanized antibodies after further affinity maturation mutation of the CDRs by yeast display.

The terms "human antibody" and "human antibody" are used interchangeably to mean that one or more variable and constant regions are derived from human immunoglobulin sequences. One preferred way is that all variable and constant regions are derived from human immunoglobulin sequences, i.e., "fully human antibodies" or "fully human antibodies". These antibodies can be prepared by a variety of means, including the construction of natural single-chain phage human antibody libraries by phage display techniques, isolation of B cells from human PBMC, spleen, lymph node tissues, or by immunization of transgenic mice expressing human antibody light and heavy chains, and screening of the resulting antibodies. The human antibodies of the present disclosure also include antibodies that still bind the antigen of interest, obtained by mutation of one or more amino acids on the basis of human antibodies.

"antibodies" of the present disclosure include, in addition to full-length antibodies, antigen-binding fragments that bind antigen. The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution). 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 by a disulfide bond at the hinge region), and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments may also be incorporated into single domain antibodies, maxiantibodies, miniantibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-. Antigen-binding fragments may also be grafted to polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. patent No. 6,703,199, which describes fibronectin polypeptide miniantibodies).

The term "scFv" or "single chain antibody" refers to a fusion protein comprising at least one antibody fragment comprising the variable region of a light chain and at least one antibody fragment comprising the variable region of a heavy chain, wherein the light 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, the scFv may have the VL and VH variable regions described in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

The portion of the CAR of the present disclosure comprising an antibody or antibody fragment thereof can exist In a variety of forms In which the antigen binding domain is expressed as part of a contiguous 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: antibody Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A Laboratory, Cold Spring Harbor, New York; Houston et al, 1988, Proc Natl.Acad.Sci.USA 85: 5879-. In one aspect, the antigen binding domain of the CAR compositions of the disclosure includes 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 which primarily contributes to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) per heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) per light chain variable region. The amino acid sequence boundaries of the CDRs may be determined using any of a variety of well known protocols, including the "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 ImMunogenetics IMGT (Lefranc M.P., immunogist, 7, 132-136 (1999); Lefranc, M.P. et Al, Dev.Comp.27, 55-77(2003), (2003) et Al, for example, following the classical Kabat numbering convention, the amino acid sequence boundaries in the variable domain (VH) are residues 35-31), the CDR 35-34 (VH) and CDR 35-34 (CDR 54), the light chain numbering convention (LCDR 3675-3650) (35-2) and the LCDR 2-34 (35-11) CDR 75 (35-3) and the LCDR2 (35-3) CDR 75 (35-3) residues in the variable domain (LCDR2), CDR amino acid numbers in VH were 26-32(HCDR1), 52-56(HCDR2) and 95-102(HCDR 3); and amino acid residues in VL are numbered 26-32(LCDR1), 50-52(LCDR2) and 91-96(LCDR 3). By combining the CDR definitions of both Kabat and Chothia, the CDRs are made up of amino acid residues 26-35(HCDR1), 50-65(HCDR2) and 95-102(HCDR3) in the human VH and amino acid residues 24-34(LCDR1), 50-56(LCDR2) and 89-97(LCDR3) in the human VL. Following the rules of IMGT, the CDR amino acid residue numbers in VH are approximately 26-35(CDR1), 51-57(CDR2) and 93-102(CDR3), and the CDR amino acid residue numbers in VL are approximately 27-32(CDR1), 50-52(CDR2) and 89-97(CDR 3). Following the IMGT rules, the CDR regions of the antibody can be determined using the program IMGT/DomainGap Align. Unless otherwise indicated, the variable regions and CDR sequences of antibodies to which embodiments of the disclosure are directed are subject to the "Kabat" numbering convention.

The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the 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, when the amino acid sequences are aligned and gaps are introduced, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. For purposes of determining percent amino acid sequence identity, alignments can be accomplished in a variety of ways that are within the skill in the art, e.g., 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 being compared.

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as the Cold spring harbor antibody protocols, chapters 5-8 and 15. For example, a mouse can be immunized with human EpCAM or a fragment thereof, and the resulting antibody can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibody or antigen-binding fragment of the invention is genetically engineered to incorporate one or more human FR regions in the CDR regions of nonhuman origin. Human FR germline sequences can be obtained from the website http:// IMGT. cities.fr of ImmunoGeneTiCs (IMGT) or from the immunoglobulin journal, 2001ISBN012441351, by aligning the IMGT human antibody variable region germline gene database with the MOE software.

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 may be cloned and recombined into a GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Stable clones were obtained by expression of antibodies that specifically bind to human EpCAM. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified by conventional techniques. For example, purification is carried out using an A or G Sepharose FF column containing a buffer adjusted. Non-specifically bound fractions are washed away. And eluting the bound antibody by using a pH gradient method, detecting the antibody fragment by using SDS-PAGE, and collecting. The antibody can be concentrated by filtration by a conventional method. 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 can spread locally or across 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 "a disease associated with the expression of mesothelin" includes, but is not limited to, diseases associated with the expression of mesothelin or conditions associated with cells expressing mesothelin, including, for example, proliferative diseases such as cancer or malignancies or pre-cancerous conditions 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 can 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 those 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 can be replaced with amino acid residues from the same side chain family, and the altered CAR can be tested for the ability to bind mesothelin, for example, using the functional assays described herein.

The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., the 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 the appropriate NK receptor or signaling domain of the 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 regulates activation of the immune cell for at least some aspect of the immune cell signaling pathway in a stimulatory manner. In one aspect, the signal is a primary signal initiated by, for example, binding of the TCR/CD3 complex to peptide-loaded MHC molecules, 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 the "primary signaling domain") that functions in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM-containing cytoplasmic signaling sequences particularly useful in the present disclosure include, but are not limited to, those derived from: CD3 ζ, the common FcR γ (FCER1G), fcyriia, FcR β (FcEpsilon R1b), CD3 γ, CD3 δ, CD3 e, CD79a, CD79b, DAP10, and DAP 12. In a specific CAR of the disclosure, the intracellular signaling domain in any one or more CARs of the disclosure includes an intracellular signaling sequence, e.g., the primary signaling sequence of CD 3-zeta. In specific CARs of the disclosure, the primary signaling sequence of CD 3-zeta is as set forth in SEQ ID NO: 79 or equivalent residues from a non-human species such as mouse, rodent, monkey, ape, etc.

By "immune effector cell" is meant a cell involved in an immune response (e.g., promoting an immune effector response). Examples of immune effector cells include T cells, such as α/β T cells and γ/δ 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 on its surface a foreign antigen complexed with a Major Histocompatibility Complex (MHC). T-cells can recognize these complexes using their T-cell receptor (TCR). The APC processes the antigen and presents it to the T-cell.

The term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of the CAR-containing cell, e.g., a CART cell. Examples of immune effector functions in, for example, CART cells include cytolytic and helper activities, 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 signals 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 costimulatory intracellular signaling domain may comprise a cytoplasmic sequence from a co-receptor or a costimulatory molecule. The primary intracellular signaling domain may include signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from: CD3 ζ, common FcR γ (FCER1G), Fc γ RIIa, FcR β (FcEpsilon R1b), CD3 γ, CD3 δ, CD3 epsilon, CD79a, CD79b, DAP10, and DAP 12.

The term "zeta" or alternatively "zeta chain", "CD 3-zeta" or "TCR zeta" is defined as a protein as provided by GenBan acc No. bag36664.1 or equivalent residues from non-human species (e.g. mouse, rodent, monkey, ape etc.) and "zeta stimulating domain" or "CD 3-zeta stimulating domain" or "TCR-zeta stimulating domain" is defined as an amino acid residue from the cytoplasmic domain of the zeta chain or a functional derivative thereof sufficient to functionally convey 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 equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) as a functional homolog thereof.

The term "co-stimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory 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, which promote an effective immune response. Costimulatory 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(CD11a/CD18), ICOS (CD278) and 4-1BB (CD 137). Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHT TR), SLAMF7, NKp80(KLRF 80), NKp80, CD160, CD80 alpha, CD80 beta, IL 280 gamma, IL7 80 alpha, ITGA 80, VLA 80, CD49 80, ITGA 80, IA 80, CD49 80, ITGA 80, VLA-6, CD49 80, ITGAD, CD11 80, ITGAE, CD103, ITGAL, CD11 80, LFA-1, ITGAM, CD11 80, ITGAX, CD11 80, IT3672, CD80, ITGB 80, CD LFA-1, ACAGN-1, ITGAMMA, CD11 80, ITGAMMA 80, CD-TASALGP, CD80, CD-TASALGB, CD-TASALG-CD-TAAMB (TARGK, CD-.

The costimulatory intracellular signaling domain may be the intracellular portion of the costimulatory 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 (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, antigen-1 associated with lymphocyte function (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind CD83, and the like.

The intracellular signaling domain may comprise the entire intracellular portion of the molecule or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

The term "4-1 BB" refers to a member of the TNFR superfamily having an amino acid sequence as provided in GenBank acc.no. aaa62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape and the like. In one aspect, the "4-1 BB co-stimulatory domain" is defined as amino acid residue 214-255 of GenBank Acc.No. AAA62478.2, or equivalent residues from a non-human species such as mouse, rodent, monkey, ape, and the like.

The term "encode" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, as a template in biological processes 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, the nucleotide sequence of which is identical to the mRNA sequence and is typically 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 encoding the protein or other product of the gene or cDNA.

Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" 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 the protein may contain introns in some forms.

The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer 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 can be used to deliver the isolated nucleic acid to the interior of a cell. A wide variety of 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 an autonomously replicating plasmid or virus. 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, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral (lentivirus) vectors, and the like.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising 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) that incorporate the recombinant polynucleotide.

The term "lentivirus" refers to a species of the retrovirus family. 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, and thus they are one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are examples of lentiviruses. The term "lentiviral vector" refers to a vector derived from at least a portion of the lentiviral genome, and specifically includes self-inactivating lentiviral vectors such as those provided in Milone et al, mol. ther.17(8): 1453-. Other examples of lentiviral vectors that may be used in the clinic include, but are not limited to, for example, the LENTIVECTOR gene delivery technology from Oxford BioMedica, the vector system from Lentigen LENTIMAX, and the like. Non-clinical varieties of lentiviral vectors are also available and will be known to those skilled in the art.

The term "isolated" refers to an alteration or removal from the native 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 separated from the coexisting materials of its natural state is "isolated. An 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 "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the 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 the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous with each other and must be in 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 joined to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (which are also commonly referred to in the art as proteins, of which there are various types). "polypeptide" includes, 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 native peptide, a recombinant peptide, or a combination thereof.

The term "promoter" refers to a DNA sequence recognized by, or introduced into, the synthetic machinery of a cell, which is required to initiate specific transcription of a polynucleotide sequence.

The term "flexible polypeptide linker" refers to a peptide linker consisting of amino acids such as glycine and/or serine residues, used alone or in combination, that links variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly)₄Ser)_nWherein n is a positive integer equal to or greater than 1. For example, n is 1, n is 2, n is 3, n is 4, n is 5, n is 6, n is 7, n is 8, n is 9, and n is 10. Also included within the scope of the present disclosure are linkers described in WO2012/138475, which are incorporated herein by reference.

The term "transfected" or "transformed" or "transduced" refers to the process by which an 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 cells of a primary subject 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.

By "pharmaceutical composition" is meant a mixture containing one or more antibodies that specifically bind MSLN, CART cells, and other chemical components described herein, such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for delivery of the antibody or antigen-binding fragment. The carrier may be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (e.g., antioxidant, antibacterial or antifungal agent), sweetener, absorption retarder, wetting agent, emulsifier, buffer, or the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

"administration," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering", "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by an agent, a diagnostic, a 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 "prevention" refers to prophylactic or protective treatment of a disease or condition.

The term "treating" refers to slowing or improving the progression, severity, and/or duration of a proliferative disorder, or improving one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder as a result of administering one or more therapies (e.g., one or more therapeutic agents such as a CAR of the invention). In certain embodiments, the term "treating" refers to ameliorating at least one measurable physical parameter of a proliferative disorder, such as tumor growth, not necessarily discernible by the patient. In other embodiments, the term "treating" refers to inhibiting the progression of a proliferative disorder, either 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 disclosure is further described below in conjunction with the following examples, which are not intended to limit the scope of the disclosure. The experimental methods of the present disclosure, in which specific conditions are not specified, are generally performed according to conventional conditions, such as the antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Examples

Example 1 preparation of anti-MSLN Single chain antibody molecule

1. Through a large number of experiments, a series of scFv (MSLN-scFv for short) which specifically binds to MSLN are constructed, and the variable regions, CDR regions and full-length sequences of the light/heavy chain are shown as follows:

TABLE 1 MSLN-scFv heavy chain variable region CDR

TABLE 2 MSLN-scFv light chain variable region CDR

2. The variable regions of the heavy and light chains of the scFv of the present disclosure against MSLN are shown below:

TABLE 3 MSLN-scFv heavy and light chain variable regions

The VH and VL described above are linked via a linker, an exemplary linker is (G4S) n, where n.gtoreq.1, to obtain a single chain antibody. Exemplary heavy and light chain variable regions of the above antibody are connected via a linker (G4S)₃And connecting to construct MSLN-scFv.

TABLE 4 MSLN-scFv heavy and light chain variable region composition

Note: H1L1 shows that the MSLN-scFv has a heavy chain variable region of H1 and a light chain variable region of L1.

Exemplary sequences of MSLN-scFv in the present disclosure are 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

SEQ ID NO：72

H10L8：

EVQLVESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIK

SEQ ID NO：73

H9L7：

QQQLEESGGGLVKPEGSLTLTCKASGFDLGFYFYACWVRQAPGKGLEWIACIYTAGSGSTYYASWAKGRFTISKASSTTVTLQMTSLAAADTATYFCARSTANTRSTYYLNLWGPGTLVTVSSGGGGSGGGGSGGGGSDVVMTQTPASVSEPVGGTVTIKCQASQRISSYLSWYQQKPGQRPKLLIFGASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSYAYFDSNNWHAFGGGTEVVV

SEQ ID NO：74

exemplary, the CDR region sequences of MSLN-scFv are as follows:

TABLE 5 CDRs of MSLN-scFv

The CDR regions of the MSLN-scFv in this disclosure have commonality and can be summarized as follows:

the light chain variable region comprises an LCDR as shown below:

LCDR1 such as X₁ASQRISSYLS (SEQ ID NO: 94);

LCDR2 such as X₂ASX₃LX₄S (SEQ ID NO: 95);

LCDR3 is shown as QSYAYFDSNNWHA (SEQ ID NO: 6); and

the heavy chain variable region comprises an HCDR as shown below:

HCDR1 such as FYX₅YAC (SEQ ID NO: 91);

HCDR2 such as CIYTAGSGSTYYAX₆X₇X₈KG (SEQ ID NO: 92); and

HCDR3 such as STX₉NTRSTYYLNX₁₀(SEQ ID NO: 93);

Expression and purification of MSLN-scFv antibodies

The end of the MSLN-scFv sequence is added with a His tag, synthesized by gene companies, and then cloned into a Phr-IgG expression vector to be expressed in a Gbico Expi-CHO-S expression system. A sample of the supernatant was collected, centrifuged at high speed to remove impurities, subjected to affinity chromatography using Capto L (GE healthcare 17-5478-02), washed with pure water, and equilibrated with PBS to the column, the supernatant was bound to the column, and the target protein was eluted at pH3.5 with 0.1M acetate buffer and neutralized with 1M Tris-HCl. And A280 detecting the absorbance to calculate the yield, thereby obtaining the MSLN-scFv antibody.

Example 2 construction of MSLN-CAR

And recombining the MSLN-scFv fragment with a transmembrane region, a signal transduction domain, a costimulatory domain and the like to construct a complete MSLN-CAR. The related sequences are shown below:

CD8 leader amino acid sequence:

MALPVTALLLPLALLLHAARP

SQE ID NO：75

CD8 hinge region amino acid sequence:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

SQE ID NO：76

amino acid sequence of 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 for the construction of the complete MSLN-CAR is 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, generating a full-length CAR molecule in a single coding frame and expressed using the EF 1a promoter.

EF1 alpha promoter

ctagtggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaacgggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgaagcttcgaggggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactctacgtctttgtttcgttttctgttctgcgccgttacagatccaagctgtgaccggcgcctac

SQE ID NO: 90 control and other protein sequences used in this 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 a negative control

RS7 antibody heavy chain:

QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：98

RS7 antibody light chain:

DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：99

example 3 third Generation of lentivirus packaging System and packaging of lentivirus vectors

The third generation of lentivirus packaging system used, including expression plasmid, envelope plasmid and packaging plasmid four plasmid system for packaging virus.

Inserting the MSLN-CAR nucleic acid sequence into the multiple cloning site of pCDH-EF1-MCS-IRES-copGFP vector (SBI, cat # CD530A-2), and deleting the IRES-copGFP sequence to obtain expression plasmid pCDH-EF1 alpha-anti-MSLN-CAR; the envelope plasmid and the packaging plasmid were pMD2.G (Youbao, cat # VT1443), pMDLgpRRE (Youbao, cat # VT1449), and pRSV-Rev (Youbao, cat # VT1445), respectively. HEK293 cells are cultured to about 80% density by using a DMEM medium and 10% FBS, expression plasmids pCDH-MSLN-CAR and three auxiliary plasmids pMDLg-pRRE, pRSV-Rev and pMD2.G are respectively mixed according to the mass ratio of 4:3:2:1, PEI reagent is adopted to transfect the HEK293 cells, supernatant is harvested after 48 hours, and the harvested lentivirus supernatant is centrifuged for 2 hours by a high-speed centrifuge of 25000g to obtain the lentivirus.

Example 4 preparation of MSLN-CART

Healthy volunteers were recruited and 100ml of blood was intravenously drawn by medical professionals into an anti-coagulation tube (BD, 367886). The blood was mixed with an equal amount of PBS buffer (containing 2% fetal bovine serum). The PBMC separation tube was taken for Sepmate-50(STEMCELL Technology 86450), 15mL of Ficoll buffer (GE healthcare, 17-5442-02) was added, and 32mL of a blood-PBS mixture was added. After centrifugation at 1200g for 10min at room temperature, the upper suspension containing PBMC is quickly poured into a clean 50mL centrifuge tube, centrifuged at 500g for 8min at room temperature, the supernatant is discarded, and the PBMC is obtained by resuspension with PBS.

Used separation kit EasySep^TMHuman T cell isolation kit (Stem cell Co., 17951) further isolated T cells from PBMC. The resulting T cells were cultured in a culture medium at 1:1 ratio of CD3/CD28 magnetic beads (Dynabeads)^TMHuman T-Activator CD3/CD28, GIBCO, 11132D), in X-VIVO15(Lonza, 04-418Q), contains rhIL-2 (R) at a final concentration of 100IU/mL&D, 202-IL-050) at a cell density of 1X 10⁶Per mL, 37 ℃, 5% CO₂Is activated under the culture conditions of (1).

After 24 hours of activation, the mixture of cells and magnetic beads was collected, centrifuged to remove the supernatant, resuspended in the above medium (X-VIVO15+100IU/mL rhIL-2), and removed at 1X 10⁶Cells were plated in 48-well plates, concentrated virus was added at an MOI of 5, and finally cell density was adjusted to 2X 10⁶Centrifuging at 2000g at 32 ℃ for 1 hour; after centrifugation, the volume ratio of 1:1 supplement medium (X-VIVO15+100IU/mL rhIL2), placed at 37 ℃ with 5% CO₂Culturing in an incubator for 3 days. After 3 days, the beads were removed and the cell suspension was placed in the magnetic pole for easy Sep^TMD Magnetite Particles (STEMCELL, 19550), after two minutes of standing, the magnetic beads on the tube wall were discarded to obtain MSLN-CART cells. The 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 culture⁵cells/mL to 2X 10⁶cells/mL。

Test example

Test example 1 binding of anti-MSLN Single-chain antibody to target cell

The hMSLN was constructed into pLVX-EF1-LucG plasmid, transfected with K562 cells (purchased from ATCC), and then monoclonal was picked to obtain K562-hMSLN-lucG cells.

Adjusting the cell density of K562-hMSLN-LucG to 3 x 10⁶Per mL, 100. mu.L per well of a 96-well round bottom plate. Washed once with PBS + 2% FBS buffer, centrifuged at 500g for 3 min and the supernatant discarded. Add 200. mu.L of the supernatant of SLN-scFv to each well, incubate at room temperature for 30 min, centrifuge at 500g for 3 min, and discard the supernatant. Washed 2 times with PBS + 2% FBS buffer, centrifuged 500g for 3 min and the supernatant discarded. Dilute the secondary antibody His-tag antibody [ iFluor 647 ] with PBS + 2% FBS buffer](genscript, A01802-100) at a concentration of 2. mu.g/mL, 100. mu.L of each sample was 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 minutes, discard supernatant. The cells were resuspended in PBS + 2% FBS buffer 100. mu.L and pipetted well and the samples were analyzed by flow cytometry (Invitrogen, Atture NxT). The flow data were analyzed by FlowJo-VX-10.4 and plotted against a Geometric Mean of iFluor 647 fluorescence intensity, the results of which 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 all MSLN-scfvs bound to K562-hMSLN-LucG cells, indicating that the anti-MSLN single chain antibodies of the present disclosure can specifically recognize human MSLN.

Test example 2 detection of affinity of anti-MSLN Single-chain antibody to antigen

Biacore was used to determine the affinity of the MSLN-scFv single chain antibody to the antigen MSLN-R3-hFc 4.

A certain amount of antigen MSLN-R3-hFc4 is subjected to affinity capture by a CM5 biosensor chip (Cat. # BR100530, GE) coupled with anti-human antibodies, and then a certain concentration of anti-MSLN-scFv single-chain antibody flows through the surface of the chip. The reaction signal was detected in real time using a Biacore T200 instrument to obtain binding and dissociation curves. After each cycle of dissociation was complete, 3M MgCl was used₂(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 data obtained were fitted to the (1:1) Langmuir model using GE Biacore T200 Evaluation version 3.0 software to obtain affinity values.

TABLE 6 MSLN-scFv single chain antibodies with affinity for MSLN-R3-hFc4

The results show that the MSLN-scFv in this disclosure has higher affinity to the MSLN-R3-hFc4 antigen.

Test example 3 MSLN-CAR mediated T cell activation Capacity assay

Firstly, JNL-MSLN-CAR cells are constructed, and the density of NFAT-Luc Reporter T Lymphocytes (InvivoGen, product number jktl-NFAT, JNL cells for short) is adjusted to 2 x 10⁶Per mL, add 100. mu.L to a 96-well round bottom plate. The MSLN-CAR lentivirus amount was calculated as MOI-5 and centrifuged at 2000g for 60 min at 32 ℃ after addition of lentivirus. Adding preheated 1640 culture medium after centrifugation to adjust the cell density to 1 × 10⁶And (4) placing the cells in an incubator for continuous culture for 4 days to obtain the JNL-MSLN-CAR cells.

On the fifth day, JJNL-MSLN-CAR cells and JNL-CAR-N2 were adjusted to a cell density of 1X 10⁶one/mL, 20. mu.L to 96-well flat-bottom plate, K562-hMSLN cell density 2.5X 10⁵ Take 80. mu.L/mL, put into 96-well flat-bottom plate with JNL-MSLN-CAR, put into incubator to culture for 18-24 hours.

QUANTI-Luc^TMPreparation of analytical reagents: a clean 50mL centrifuge tube was taken and 25mL sterile water was added. Mixing QUANTI-Luc^TM(InvivoGen, rep-qlc2) the whole powder was poured into a jar and dissolved, and stored at 4 ℃ in the dark for one week.

The cells were removed from the incubator, centrifuged at 1000rpm for 5 minutes, 20. mu.l of culture supernatant was aspirated into a 96-well plate, and 50. mu.L of QUANTI-Luc was added^TMThe reagents were analyzed and mixed by shaking for 5 seconds. Immediately after mixing, bioluminescence was measured using a microplate reader, and the results 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 were specifically activated by K562-hMSLN. And the extent of JNL-MSLN-CAR cell activation is related to the scFv sequence of CAR, with JNL-MSLN-CAR cells comprising H6L6-CAR being the best performing.

Test example 4 detection of culture Activity Rate, proliferative Capacity and Positive Rate of CAR expression of MSLN-CART cells

(1) Detecting the culture survival rate and proliferation capacity of the CART cells:

and gently beating and uniformly mixing each group of cells of the MSLN-CART by using a pipette, taking 20 mu L to 1.5mL of a centrifuge tube, uniformly mixing with 20 mu L of diluted Trypan Blue Solution (0.1 percent, Gibco, 15250061), sucking 20 mu L of the mixture to a cell counting plate, reading the cell density and the cell viability by using a cell counter, and calculating the total number and the proliferation times of the cells according to the culture volume. Wherein:

cell survival rate was dead cells/(live cells + dead cells) × 100%.

Fold of 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 groups of cells 5X 10⁵Centrifuging the cells at 500g for 3 minutes, and discarding the supernatant; 1mL of buffer (PBS + 0.5% BSA) was added for resuspension, 500g, centrifugation was performed for 3 minutes, and the supernatant was discarded and resuspended in 100. mu.L of buffer. Adding 2 mu L of primary antibody of CD19(FMC63 antibody) into N2-CART cells, adding 2 mu L of MSLN Fc protein into each group of MSLN-CART cells, mixing uniformly, and incubating for 30 minutes at 4 ℃; after one wash with buffer, secondary antibody was added, wherein 2. mu.L of PE goat-anti-mouse, IgG (Biolegend, 554061), 2. mu.L of PE anti-human Fc, IgG (Biolegend, 405307) secondary antibody was added to each group of N2-CART cells, and incubated at 4 ℃ for 30 minutes. After incubation, the cells were washed 2 times with buffer, 500. mu.L of buffer was added to resuspend the cells, mixed 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: d represents day.

TABLE 8 fold proliferation of MSLN-CART

Note: d represents day.

TABLE 9 CAR positivity vs MFI data Table

Test example 5 repeated antigen stimulation of CART cell proliferation potency assay

1. First round of stimulation (days 1-4):

resuscitation of target cells SKOV3 (purchased from ATCC, DMEM medium + 10% fetal bovine serum, purchased from Thermo Fisher, 10566016), starting cell density 2X 10⁵one/mL, cultured to logarithmic growth phase. Part of the target cells (remaining normal cultures) were resuspended in X-VIVO medium (IL-2-free, Lonza, 04418Q) and plated in 48-well plates (1X 10)⁵One/well), placed in a 37 degree incubator, control wells are not plated with target cells. Effector cells (MSLN-CART) required for the experiment were washed once with PBS, counted after resuspension in X-VIVO medium (without IL-2), and adjusted with T cells that were not transfected with CAR to achieve consistent total cell mass for each group of effector cells. After the target cells were attached, the supernatant was aspirated, effector cells were added to 48-well plates at an effector-to-target ratio E: T ratio of 1:1, 3 wells per group, and X-VIVO medium (no IL-2) was supplemented to 600 μ L/well. Effector cells were incubated with target cells for 2 days and then supplemented with 300. mu. L X-VIVO medium (without IL-2) per well. Co-incubationAfter 4 days, cells in the supernatant of the co-incubation system were collected, resuspended in 1ml of X-VIVO medium (without IL-2) after centrifugation and counted to calculate the fold expansion, where:

first round fold expansion is the amount of cells after co-incubation/amount of cells initially added.

2. Second round stimulation (days 4-8):

normal cultured target cells were resuspended in X-VIVO medium (without IL-2) and plated in 48-well plates (1X 10)⁵One per well), placed in a 37-degree incubator (no target cells plated in control wells), after the cells adhere, effector cells are added to 48-well plates according to the counting result in the ratio of effector cells to target cells E: T ratio 1:1, 3 wells per group, and supplemented with X-VIVO medium (no IL-2) to 600 μ L per well. Effector cells were incubated with target cells for 2 days and then supplemented with 300. mu. l X-VIVO medium (without IL-2) per well. After 4 days of co-incubation, cells in the supernatant of the co-incubation system were collected, resuspended in 1ml of X-VIVO medium (no IL-2) after centrifugation and counted to calculate the fold expansion, where:

second round of fold expansion ═ first round fold expansion x cell mass after co-incubation/initial cell mass added

3. Third round of stimulation (days 8-12):

performing a third round of stimulation according to the second round of stimulation method, wherein:

third round of amplification fold ═ first round of amplification fold × second round of amplification fold × cell mass after co-incubation/initial cell mass added.

The results of the measured CAR-T cell proliferation are shown in FIGS. 4A-4B.

From the results, the number of the cells stimulated by the SKOV3 cells is remarkably increased compared with the MSLN-CART cells without antigen stimulation, which indicates that MSLN single-chain antibodies in the anti-MSLN-CART can effectively recognize MSLN antigens and activate T cell expansion. In particular, H6L6-CAR, H6L4-CAR, and H5L3-CAR significantly stimulated T cell expansion.

Test example 6 detection of killing ability of MSLN-CART cells against target cells in vitro

After SK-OV-3 cells (purchased from ATCC) were transfected with pLVX-EF1-LucG plasmid containing lucifrase and GFP, a single clone was selected to obtain the SK-OV-3-LucG cell line.

The density of target cells (SK-OV-3-LucG) is adjusted to 5 × 10⁴Per mL, 100. mu.L of the suspension was centrifuged at 600rpm for 2 minutes in a 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 without phenol red (Lonza, 04-418Q) was added to each well. The ratio of effector cells (MSLN-CART) to target cells E: adding effector cells with T ratio (20: 1-0.625: 1), and mixing by blowing and sucking. Centrifuge at 1000rpm for 2 minutes and incubate for 16 hours. Centrifuge at 300g for 5 min and discard the supernatant. Wash once with PBS and discard supernatant. 100. mu.L of PBS was added, and 100. mu.L of a detection solution (Promega corporation, Ltd.)

Luciferase Assay System, E2520), incubation for 10-20 minutes at room temperature, detection of bioluminescence values using a microplate reader, and calculation of the killing rate.

The killing rate is (reading of target cells alone-reading after addition of effector cells)/(reading of target cells alone-reading of medium) × 100%.

The results of the killing are shown in table 10 and fig. 5.

TABLE 10 kill rate of MSLN-CART cells against target cells

The results show that the MSLN-CART cell has obvious killing capacity on SK-OV-3-LucG cells, while N2 does not kill SK-OV-3-LucG cells, which indicates that the MSLN-CART cell can specifically kill MSLN positive cells.

Test example 7 in vitro killing of target cell cytokine secretion by MSLN-CART

The cell supernatants of MSLN-CART after co-incubation with target cells (E: T1: 1, E: T5: 1) in test 6 were assayed for IL-2 and IFN- γ concentrations in their supernatants using human IL-2ELISA Kit II (BD, 550611) and human IFN- γ ELISA Kit II (BD, 550612), and the results are shown in fig. 6A-6B.

The results show that candidate cells all produced significant amounts of IFN- γ and IL-2 cytokines when co-cultured with target cells, suggesting that MSLN-CART cells can both exhibit characteristics of killer T cells.

Test example 8 evaluation of drug efficacy in MSLN-CART cell mouse transplanted tumor model in vivo

NDG mice (purchased from paoysai corporation), female, 6-8 weeks, breeding environment: SPF grade. One week after acclimation, mice were randomly divided into 6 groups of 6-8 mice each. 5X 10 per mouse⁶SKOV3 tumor cells were inoculated subcutaneously into right scapular region of the right shoulder for molding, and 18 days later, the cells were randomly divided according to the average tumor size, and each mouse was reinfused with 2.5X 10 cells⁷The weight and the tumor size of the mice are recorded for 2-3 times per week by each MSLN-CART cell, and the killing conditions of different CARTs on SK-O-V3 tumor cells in vivo are compared. Grouping of NDG mice and reinfusion of CART cells is as follows:

TABLE 11 mouse grouping

Grouping	Reinfused cell species	Number of cells returned	Number of mice
				1	H5L5-CAR	2.5×10⁷	8
2	H6L5-CAR	2.5×10⁷	8
				3	H6L6-CAR	2.5×10⁷	8
4	H11L8-CAR	2.5×10⁷	8
				5	N2	2.5×10⁷	7
6	PBS	Is free of	6

The results of body weight and tumor size after modeling and reinfusion of MSLN-CART cells in mice are shown in FIGS. 7A-7B.

The results show that the body weight of the mice has no obvious change after the return transfusion of the CART cells, which indicates that the mice have good tolerance to the CART cells. The in vivo anti-tumor effects of the H5L5-CAR, the H6L5-CAR, the H6L6-CAR and the H11L8-CAR are similar, and the in vivo SK-O-V3 tumor cells can be effectively killed.

Test example 9 pharmacokinetic testing of MSLN-CART cells in transplanted tumor mice

1. Proliferation assay for CART cells in mice

NDG mice (purchased from paoysai corporation), female, 6-8 weeks, breeding environment: SPF grade. One week after acclimation, mice were randomly divided into 6 groups of 7-8 mice each. 5X 10 per mouse⁶Subcutaneous inoculation and molding are carried out on the right scapular part of the SK-O-V3 tumor cell, and tumors are planted according to the average number after 18 daysSize, random grouping, 2.5X 10 reinfusion per mouse⁷An MSLN-CART cell. After the return transfusion of CART cells, on days 0 (3 hours after the return transfusion), 3, 7, 14, 21 and 28, mouse blood was collected from the orbit, and the amount of CART cells in the peripheral blood of the mice was measured, and the specific groups are shown in table 12. The number of MSLN-CART cells in the peripheral blood of mice within 4 weeks after reinfusion and the results are shown in figure 8.

TABLE 12 mouse grouping

As can be seen from the results in FIG. 8, after the mice were modeled with SK-O-V3 tumor cells, MSLN-CART cells were significantly amplified in the mice after the injection of MSLN-CART cells, but the number of N2-CART cells did not significantly proliferate, indicating that the MSLN-CART cells were specifically stimulated by SK-O-V3 tumor cells in the mice, thereby obtaining amplified signals and proliferated in large quantities.

Sequence listing

<110> Hengrui pharmaceutical Co., Ltd of Jiangsu

Shanghai Hengrui pharmaceuticals, Inc

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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> amino acid sequence of CD8 transmembrane region

<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 sequences

<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 sequences

<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 general 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

1. A Chimeric Antigen Receptor (CAR) molecule comprising:

i) (ii) an anti-mesothelin binding domain,

ii) a transmembrane domain, and

iii) an intracellular signaling domain;

LCDR1 such as X₁ASQRISSYLS (SEQ ID NO: 94);

LCDR2 such as X₂ASX₃LX₄S (SEQ ID NO: 95);

LCDR3 is shown as QSYAYFDSNNWHA (SEQ ID NO: 6); and

wherein the heavy chain variable region comprises an HCDR as shown below:

HCDR1 such as FYX₅YAC (SEQ ID NO: 91);

HCDR2 such as CIYTAGSGSTYYAX₆X₇X₈KG (SEQ ID NO: 92);

HCDR3 such as STX₉NTRSTYYLNX₁₀(SEQ ID NO: 93);

2. The CAR molecule of claim 1, wherein the anti-mesothelin binding domain comprises a light chain variable region and a heavy chain variable region, wherein:

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 1. SEQ ID NO: 10 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 3; preferably, wherein said anti-mesothelin binding domain comprises:

a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 1. SEQ ID NO: 2 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 12; or

a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 7. SEQ ID NO: 2 and SEQ ID NO: 3 HCDR1, HCDR2 and HCDR 3.

3. The CAR molecule of claim 2, wherein the anti-mesothelin binding domain comprises a light chain variable region and a heavy chain variable region, wherein:

the sequence of the light chain variable region is shown as SEQ ID NO: 25. 26, 27, 28, 29, 30 or 32; and/or

The sequence of the heavy chain variable region is shown as SEQ ID NO: 16. 17, 18, 19, 20, 21, 22, 23, 24, 31 or 33.

4. The CAR molecule of claim 2, wherein the anti-mesothelin binding domain comprises a light chain variable region and a heavy chain variable region, wherein:

a1) the sequence of the light chain variable region is shown as SEQ ID NO: 25, or has at least 95% sequence identity thereto; and/or

The sequence of the heavy chain variable region is shown as SEQ ID NO: 16. 19, 20, 21 or 22, or a variant of SEQ ID NO: 16. 19, 20, 21 or 22, respectively, have at least 95% sequence identity;

b1) the sequence of the light chain variable region is shown as SEQ ID NO: 26, or has at least 95% sequence identity thereto; and/or

The sequence of the heavy chain variable region is shown as SEQ ID NO: 16. 18, 19, 20, 21, 22, 23 or 33, or a variant of SEQ ID NO: 16. 18, 19, 20, 21, 22, 23, or 33, respectively, have at least 95% sequence identity;

c1) the sequence of the light chain variable region is shown as SEQ ID NO: 27, or has at least 95% sequence identity thereto; and/or

The sequence of the heavy chain variable region is shown as SEQ ID NO: 16. 18, 19, 20, 21, 22 or 23, or a variant of SEQ ID NO: 16. 18, 19, 20, 21, 22, or 23, respectively, have at least 95% sequence identity;

d1) the sequence of the light chain variable region is shown as SEQ ID NO: 28, or has at least 95% sequence identity thereto; and/or

e1) the sequence of the light chain variable region is shown as SEQ ID NO: 29, or has at least 95% sequence identity thereto; and/or

The sequence of the heavy chain variable region is shown as SEQ ID NO: 16. 18, 19, 20, 21, 22, 23 or 24, or a variant of SEQ ID NO: 16. 18, 19, 20, 21, 22, 23, or 24, respectively, have at least 95% sequence identity;

f1) the sequence of the light chain variable region is shown as SEQ ID NO: 30, or has at least 95% sequence identity thereto; and/or

The sequence of the heavy chain variable region is shown as SEQ ID NO: 19. 20, 21, 23 or 24, or a variant of SEQ ID NO: 19. 20, 21, 23 or 24, respectively, have at least 95% sequence identity; or

g1) The sequence of the light chain variable region is shown as SEQ ID NO: 32, or has at least 95% sequence identity thereto; and/or

The sequence of the heavy chain variable region is shown as SEQ ID NO: 31, or has at least 95% sequence identity thereto; preferably, the first and second electrodes are formed of a metal,

the sequence of the light chain variable region is shown as SEQ ID NO: 29 and the sequence of the heavy chain variable region is as shown in SEQ ID NO: 20 is shown in the figure; or

The sequence of the light chain variable region is shown as SEQ ID NO: 30 and the sequence of the heavy chain variable region is shown as SEQ ID NO: shown at 21.

5. The CAR molecule of any one of claims 1 to 4, wherein the anti-mesothelin binding domain is an scFv.

6. The CAR molecule of claim 5, wherein the scFv comprises a heavy chain variable region comprising SEQ ID NO:

as shown in SEQ ID NO: 25. 26, 27, 28, 29, 30, or 32, and/or

As shown in SEQ ID NO: 16. 17, 18, 19, 20, 21, 22, 23, 24, 31, or 33; preferably, wherein said scFv comprises:

7. The CAR molecule of any one of claims 1 to 6, wherein the anti-mesothelin binding domain comprises the amino acid sequence as set forth in SEQ ID NO: 34-74; preferably, the anti-mesothelin binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 51. 52, 53, 58, 59 or 60.

8. The CAR molecule of any one of claims 1 to 7, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from any one of: the α, β or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154.

9. The CAR molecule of claim 8, wherein the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 77.

10. The CAR molecule of any one of claims 1 to 9, wherein the anti-mesothelin binding domain is connected to the transmembrane domain by a hinge region.

11. The CAR molecule of claim 10, wherein the hinge region comprises a sequence as set forth in SEQ ID NO: 76, respectively, and a sequence shown in fig. 76.

12. The CAR molecule of any one of claims 1 to 11, wherein the intracellular signaling domain is a functional signaling domain of CD3 ζ; preferably, wherein said functional signaling domain of CD3 ζ comprises a sequence as set forth in SEQ ID NO: 79, or a pharmaceutically acceptable salt thereof.

13. The CAR molecule of any one of claims 1 to 12, wherein the intracellular signaling domain further comprises a co-stimulatory domain.

14. The CAR molecule of claim 13, wherein the co-stimulatory domain comprises a functional signaling domain selected from any of the following proteins: OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1, CD278, and 4-1 BB.

15. The CAR molecule of claim 13, wherein the co-stimulatory domain is a functional signaling domain of 4-1 BB; preferably, it comprises the amino acid sequence as set forth in SEQ ID NO: 78, or a sequence shown in seq id no.

16. The CAR molecule of any one of claims 1 to 15, wherein the intracellular signaling domain comprises the amino acid sequence set forth as SEQ ID NO: 78 and/or the sequence as shown in SEQ ID NO: 79, or a pharmaceutically acceptable salt thereof.

17. The CAR molecule of any one of claims 1 to 16, further comprising a leader sequence.

18. The CAR molecule of claim 17, wherein the leader sequence comprises the sequence set forth as SEQ ID NO: 75, or a sequence shown in seq id no.

19. The CAR molecule of any one of claims 1 to 18, comprising the amino acid sequence set forth as SEQ ID NO: 80-87, preferably comprising the sequence shown in SEQ ID NO: 82 or 85, respectively.

20. An antibody that specifically binds mesothelin, comprising a light chain variable region and a heavy chain variable region, wherein:

the heavy chain variable region comprises the amino acid sequences shown as SEQ ID NO: 1. SEQ ID NO: 10 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 3; preferably, wherein said antibody that specifically binds mesothelin comprises:

21. The antibody of claim 20, comprising a light chain variable region and a heavy chain variable region, wherein:

the sequence of the light chain variable region is shown as SEQ ID NO: 25. 26, 27, 28, 29 or 30; and/or

The sequence of the heavy chain variable region is shown as SEQ ID NO: 16. 17, 18, 19, 20, 21, 22, 23 or 24.

22. The antibody of claim 21, which specifically binds mesothelin, comprising a light chain variable region and a heavy chain variable region, wherein:

a1) the sequence of the light chain variable region is shown as SEQ ID NO: 25, or has at least 95% sequence identity thereto; and

b1) the sequence of the light chain variable region is shown as SEQ ID NO: 26, or has at least 95% sequence identity thereto; and

the sequence of the heavy chain variable region is shown as SEQ ID NO: 16. 18, 19, 20, 21, 22 or 23, or a variant of SEQ ID NO: 16. 18, 19, 20, 21, 22, or 23 have at least 95% sequence identity;

c1) the sequence of the light chain variable region is shown as SEQ ID NO: 27, or has at least 95% sequence identity thereto; and

d1) the sequence of the light chain variable region is shown as SEQ ID NO: 28, or has at least 95% sequence identity thereto; and

e1) the sequence of the light chain variable region is shown as SEQ ID NO: 29, or has at least 95% sequence identity thereto; and

the sequence of the heavy chain variable region is shown as SEQ ID NO: 16. 18, 19, 20, 21, 22, 23 or 24, or a variant of SEQ ID NO: 16. 18, 19, 20, 21, 22, 23, or 24 have at least 95% sequence identity; or

f1) The sequence of the light chain variable region is shown as SEQ ID NO: 30, or has at least 95% sequence identity thereto; and

the sequence of the heavy chain variable region is shown as SEQ ID NO: 19. 20, 21, 23 or 24, or a variant of SEQ ID NO: 19. 20, 21, 23 or 24 have at least 95% sequence identity; preferably, the first and second electrodes are formed of a metal,

23. The antibody that specifically binds mesothelin according to claim 22, which is a single chain antibody; preferably, wherein said single chain antibody comprises:

as shown in SEQ ID NO: 16. 17, 18, 19, 20, 21, 22, 23 or 24, and

as shown in SEQ ID NO: 25. 26, 27, 28, 29 or 30.

24. The antibody that specifically binds mesothelin according to any one of claims 20 to 23, comprising the amino acid sequence set forth as SEQ ID NO: 34-72; preferably, the antibody that specifically binds mesothelin comprises an amino acid sequence as set forth in SEQ ID NO: 51. 52, 53, 58, 59 or 60.

25. A nucleic acid molecule encoding the CAR molecule of any one of claims 1 to 19 or the antibody of any one of claims 20 to 24 that specifically binds mesothelin.

26. The nucleic acid molecule of claim 25, comprising the sequence set forth as SEQ ID NO: 88 or 89.

27. A recombinant vector comprising the nucleic acid molecule of claim 25 or 26; preferably, wherein said recombinant vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector and retroviral vector.

28. The recombinant vector of claim 27, further comprising a promoter; preferably, wherein said promoter is the EF-1 promoter; more preferably, wherein the EF-1 promoter comprises the amino acid sequence as set forth in SEQ ID NO: 90, or a sequence shown in seq id no.

29. A cell comprising the recombinant vector of claim 27 or 28.

30. The cell of claim 29, wherein the cell is an immune effector cell and expresses a CAR molecule of any one of claims 1 to 19; preferably, wherein said immune effector cell is an NK cell, macrophage, dendritic cell or T cell; more preferably, wherein said immune effector cell is a CD8+ T cell or a CD4+ T cell.

31. A method of producing a cell comprising the step of transducing the recombinant vector of claim 27 or 28 to an immune effector cell.

32. A pharmaceutical composition comprising a therapeutically effective amount of an antibody that specifically binds mesothelin according to any one of claims 20 to 24, or a nucleic acid molecule according to claim 26 or 27, or a cell according to claim 29 or 30, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

33. Use of a cell expressing a CAR according to any one of claims 1 to 19, or an antibody according to any one of claims 20 to 24 which specifically binds mesothelin, or a nucleic acid molecule according to claim 26 or 27, or a pharmaceutical composition according to claim 32, in the manufacture of a medicament for the treatment or prevention of cancer; preferably, wherein said cancer is selected from: mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, cholangiocarcinoma, breast cancer, malignant pleural mesothelioma, non-small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, ductal adenoma of the pancreas, ovarian cancer, colorectal cancer, breast cancer, and bladder cancer; more preferably, wherein said cancer is associated with mesothelin expression.