CN112661854B - Bispecific antibody for resisting PD-L1 and TIGIT as well as preparation and application thereof - Google Patents

Abstract

The present disclosure provides bispecific antibodies that specifically bind to both human PD-L1 and TIGIT antigens. In embodiments, the bispecific antibody comprises heavy and light chain domains linked to an ScFv at the N-or C-terminus of the heavy chain. Wherein the IgG domain has binding specificity for a first antigen and the scFv portion has binding specificity for a second antigen, the first and second antigens being human PD-L1 and human TIGIT, respectively. The bispecific antibody provided by the application can simultaneously have obvious 2 ligand-receptor binding inhibition abilities, simultaneously block the binding between PD1 and PD-L1 and between CD155 and TIGIT, and restore the functions of T cells. Has relevant application prospect in the field of tumor treatment and prevention.

Description

Bispecific antibody for resisting PD-L1 and TIGIT as well as preparation and application thereof

Technical Field

The application relates to a genetic engineering product, belongs to the technical field of biomedical engineering, and in particular relates to a PD-L1 and TIGIT bispecific antibody, and preparation and application thereof.

Background

The PD-1/PD-L1 signaling pathway plays an important role in regulating immune tolerance, microbial infection and tumor immune escape. PD-1 (programmed cell death, programmed cell death factor 1) is mainly expressed in immune cells such as T cells, while PD-L1, a ligand of PD-1, is mainly highly expressed in many human tumor tissues. Targeting immune checkpoint molecules such as PD-1 or ligand PD-L1 can increase existing tumor treatment modalities, blocking PD-1/PD-L1 signaling pathways can activate inhibited T cells, thereby attacking cancer cells. At present, the expression of PD-L1 protein is detected in various human tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, melanoma and the like, and the expression level of PD-L1 is closely related to the clinic and prognosis of patients.

Although PD-L1 is expressed in a variety of tumors, less than 50% of tumor patients respond to treatment with monoclonal antibodies directed against PD-1/PD-L1, thus targeting other immune checkpoints, such as TIGIT, an immune checkpoint that may have a synergistic effect with PD-1/PD-L1, can increase and improve the use of monoclonal antibodies in tumor therapy.

TIGIT (T cell immunoglobulin and ITIM domain) is a novel expressed immunoglobulin superfamily member that is predominantly expressed on regulatory T cells (Treg), depleting cd8+ T cells, memory T cells, and Natural Killer (NK) surfaces (Stengel KF, et a1 (2012) Proc Natl Acad Sci usa.109). TIGIT consists of an immunoglobulin variable region (IgV), a transmembrane region, and a receptor tyrosine inhibitory motif (immunoreceptor tyrosine-based inhibitor motif, ITIM), is a co-inhibitory receptor protein that binds to the co-stimulatory molecule CD226 with the common ligand PVR/CD 155. TIGIT produces immunosuppressive signals by binding to the ligand CD155 or CD122, inhibiting T cell responses and NK cell killing. Normally, inhibition signals generated by high affinity TIGIT/PVR interactions may prevent killing of (self) cells that normally express PVR. However, this also occurs in tumor cells expressing PVR, which can also protect themselves from immune cell-mediated killing by inhibiting the anti-tumor response of NK cells (Stanietsky et al (2009) proc. Nat' l acad. Sei. (USA) 106:17858;Lozano eta1. (2012), J.I mmunol. 188:3869). There are studies that found TIGIT to be highly enriched on tumor-infiltrating Treg cells. Tigit+ Treg cells in tumor tissue exhibit a highly active and suppressive Treg cell phenotype. Experiments have shown that CD155 is overexpressed on melanoma cells (Inozume et al (2014) J. Invest, derma to1.134:S121-Abstract 693) and on various other tumors.

It is contemplated that tumors deplete cd8+ T cells through the PD-1/PD-L1 signaling pathway, thereby enhancing the ability to escape. Partial inhibition of NK cell killing can also be achieved through TIGIT/CD155 signaling pathway, thus synergistically blocking PD-1/PD-L1 and TIGIT/CD155 has a synergistic antitumor effect in theory. Clinical data show that the tumor inhibition effect of the TIGIT blocking antibody Tiragolumab combined with the anti-PD-L1 monoclonal antibody Atezolizumab on the second-phase clinic (NCT 03563716) is obviously better than the effect of the single drug Atezolizumab.

The bispecific antibody consists of the rabbit humanized antibody and the fully human antibody, can target PD-L1 and TIGIT 2 targets specifically at the same time, and has high-efficiency anti-tumor biological activity verified in-vitro experiments, and compared with the combined use, the bispecific antibody has great advantages in curative effect and cost.

Disclosure of Invention

The application forms a plurality of bispecific antibodies capable of simultaneously and specifically combining PD-L1 and TIGIT 2 antigens through the existing humanized anti-PD-L1 monoclonal antibody and fully humanized anti-TIGIT monoclonal antibody and in the 2 structural composition forms shown in figure 1. The bispecific antibodies (the application comprises 11, specifically, TPAmutC1, TPAmutC2, TPAmutC3, TPAmutC4, TPA5mutC1, TPA5mutC2, TPA5mutC3, TPA5mutC4, TPC1, TPCmutC2, TPCmutC 3) all have blocking activity against PD1/PD-L1, TIGIT/CD 155.

The application discloses an amino acid sequence of a bispecific antibody comprising an IgG heavy chain, a light chain, two scFv moieties linked to the C-or N-terminus of the heavy chain, a Linker that has been linked between the scFv and the IgG.

The application discloses 2 mutation points in the bispecific antibody scFv, VH44 and VL100. Cysteine is introduced between VH and VL of a single chain antibody, such that disulfide bonds are formed between VH and VL. This method is well known in the art, see for example US patent application US5,747,654; rajagopal et al, prot.Engine.10 (1997) 1453-1459; reiter et al Nature Biotechnology 14 (1996) 1239-1245; reiter et al Protein Engineering 8 (1995) 1323-1331; webber et al; which is incorporated herein by reference.

A bispecific antibody comprising two single chain antibody scFv portions having heavy and light chain IgG domains, linked to the N-or C-terminus of the heavy chain, and a linker portion connecting between scFv and IgG domains; the scFv domain has binding specificity for a first antigen and the IgG domain has binding specificity for a second antigen; the first antigen and the second antigen are different and are respectively and independently selected from PD-L1 or TIGIT; the bispecific antibody can specifically block the binding of the antigen PD-L1 and PD-1, and can simultaneously specifically block the binding of the antigen TIGIT and CD 155.

Further, the two single chain antibody scFv portions are linked to the C-terminus of the heavy chain.

Further, the first antigen is TIGIT or PD-L1, and the second antigen is PD-L1 or TIGIT.

Further, the two single chain antibody scFv portions are linked to the N-terminus of the heavy chain.

Further, the first antigen is TIGIT or PD-L1, and the second antigen comprises PD-L1 or TIGIT.

Further, the bispecific antibody is an isolated monoclonal antibody.

Further, the linker moiety linking the scFv and the IgG domain is an amino acid sequence, including but not limited to a linker fragment having an amino acid sequence of SEQ ID No. 32-SEQ ID No. 38 or having a sequence at least 98% similar to SEQ ID No. 32-SEQ ID No. 38.

Further, the linker moiety linking the scFv to the IgG domain is an amino acid sequence comprising an amino acid sequence having SEQ ID nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or an amino acid sequence having at least 98% similarity to SEQ ID nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24.

Further, the bispecific antibody has a first binding specificity for TIGIT and a second binding specificity for PD-L1.

Further, the bispecific antibody has a first binding specificity for PD-L1 and a second binding specificity for TIGIT.

Further, the bispecific antibody is selected from one or more of a fully human antibody, a humanized antibody, a chimeric antibody or a recombinant antibody.

Further, the IgG domain comprises an IgG1 constant region having the amino acid sequence of SEQ ID No. 31 or having an amino acid sequence at least 98% similar to SEQ ID No. 31.

An IgG1 heavy chain comprising an amino acid sequence having SEQ ID nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or an amino acid sequence having at least 98% similarity to SEQ ID nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24.

A kappa light chain comprising the amino acid sequence of SEQ ID nos. 4, 26 or an amino acid sequence having at least 98% similarity to SEQ ID nos. 4, 26.

A single chain antibody scFv portion that binds to PD-L1, the single chain antibody scFv portion that binds to PD-L1 comprising the amino acid sequence of SEQ ID No. 28 or an amino acid sequence having at least 95% similarity to SEQ ID No. 28.

A single chain antibody scFv portion that binds TIGIT comprising the amino acid sequence of SEQ ID No. 30 or an amino acid sequence having at least 95% similarity to SEQ ID No. 30.

An isolated nucleic acid encoding the bispecific antibody of claim 1, the IgG1 heavy chain of claim 17, the kappa light chain of claim 18, the single chain scFv portion of claims 19 and 20.

An expression vector comprising the isolated nucleic acid of claim 21.

Further, the expression vector is capable of being expressed in a cell line.

A host cell comprising the nucleic acid of claim 21.

A host cell comprising the expression vector of claim 22.

Further, the host cell is a prokaryotic cell or a eukaryotic cell.

A method of producing a bispecific antibody, the method comprising culturing the expression vector of claim 22 or 23, or comprising culturing the host cell of any one of claims 24-26, from which the bispecific antibody is expressed.

An immunoconjugate comprising the bispecific antibody of claim 1 and a cytotoxic drug.

Further, the immunoconjugate is a cytotoxic drug, including a chemotherapeutic agent, a growth inhibitory agent, a toxin, or a radioisotope.

A pharmaceutical composition comprising the bispecific antibody of claim 1, or comprising the vector or set of vectors of claim 22, or comprising the immunoconjugate of claim 28, and a pharmaceutically acceptable carrier and a second therapeutic agent, the second therapeutic agent being an agent for use in radiation therapy, chemotherapy, targeted therapy, gene therapy, immunotherapy, hormonal therapy, angiogenesis inhibition, palliative therapy, surgery, or a combination thereof.

A kit comprising the bispecific antibody of claim 1 or the pharmaceutical composition of claim 30.

Use of a pharmaceutical composition according to claim 30 for the preparation of

In one embodiment, the bispecific antibody is an isolated monoclonal antibody.

In one embodiment, the bispecific antibody may be a humanized antibody, chimeric antibody, fully human antibody, or recombinant antibody.

In one embodiment, the application discloses an expression vector comprising an expression bispecific antibody.

In one embodiment, the expression vector may be expressed in a cell.

In one embodiment, the application discloses a host cell.

In one embodiment, the application discloses a method of producing an antibody comprising culturing a host cell provided thereby to produce the antibody.

Drawings

FIG. 1 shows the composition of exemplary bispecific antibodies targeting PD-L1 and TIGIT antigens;

FIG. 2 shows an exemplary bispecific antibody ELISA method for detecting binding to PD-L1 antigen;

FIG. 3 shows an exemplary bispecific antibody ELISA method for detecting binding to TIGIT antigen;

FIG. 4 shows binding of exemplary bispecific antibodies to a CHO cell line surface expressed PD-L1 antigen;

FIG. 5 shows the binding of three bispecific antibodies to a CHO cell line surface expressed TIGIT antigen;

FIG. 6 shows the binding of four bispecific antibodies to a CHO cell line surface expressed TIGIT antigen;

FIG. 7 results of a flow cytometer of the ability of three bispecific antibodies to block interactions between PD-1 and PD-L1;

FIG. 8 results of a flow cytometer of the ability of three bispecific antibodies to block interactions between PD-1 and PD-L1;

FIG. 9 depicts flow cytometer assay results assessing the ability of representative bispecific antibodies to block interactions between TIGIT and CD 155;

FIG. 10 depicts the results of evaluating signal assays of representative bispecific antibodies in affecting the CD155/TIGIT and PD-1/PD-L1 pathways;

FIG. 11 shows the purity of representative four bispecific antibodies measured on HPLC-SEC;

FIG. 12 shows the purity of representative four bispecific antibodies measured on HPLC-SEC;

FIG. 13 shows the results of a PD1/PD-L1/TIGIT three-humanized mouse model for an exemplary antibody.

Detailed Description

The following detailed description of the application is provided for exemplary and explanatory purposes only and should not be construed as limiting the scope of the application, as it is intended to provide a better understanding of the application as defined by the appended claims.

The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. The following examples are therefore only illustrative of the application and should not be construed as limiting the scope of the application. The technical conditions not specified in the examples are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The apparatus used did not identify the manufacturer and was a conventional product available for commercial purchase.

The present application relates to bispecific antibodies that specifically bind to PD-L1 and TIGIT, in some embodiments, the bispecific antibodies comprise a first arm that binds to PD-L1 and a second arm that binds to TIGIT, in some embodiments, the bispecific antibodies comprise a first arm that binds to TIGIT and a second arm that binds to PD-L1. Examples of arm-containing domains include, but are not limited to, fab and scFv domains. The Fc domain may be IgG1, igG2, igG3, igG4 or a corresponding engineered subtype.

Examples

Embodiment case 1: expression and purification of bifunctional antibodies

Transient transfection expression: the light and heavy chain cDNA sequences of the bispecific antibody were cloned into pcDNA3.4 vector (purchased from Invitrogen) to obtain pcDNA-Bsab-H and pcDNA-Bsab-L plasmids, respectively. Recombinant plasmids were extracted using a plasmid miniprep kit (purchased from Axygen), and 293F cells were co-transfected with the extracted plasmids using a transfection reagent. The cells were cultured for seven days, the culture solution was centrifuged to collect the supernatant, loaded onto MabSelect SuRe LX column, the target antibody protein was eluted with eluent of PH3.0 and neutralized to PH7.0, and the purified antibody protein was concentrated to PBS by ultrafiltration.

Stable transfection expression: and respectively cloning the light and heavy chain cDNA sequences of the bispecific antibody into a DN2-UCOE-HL vector to obtain DN 2-UCOE-Bsa-HL plasmid. Recombinant plasmids were extracted and endotoxin removed using a degerminal miniprep kit (available from Axygen), the extracted plasmids were digested tangentially with PvuI enzyme (available from sameifex), the linearized plasmids were transfected into CHO-S cell lines and pressure-screened with G418 resistance. The cells after pressure screening are plated into 96-well plates, and the monoclonal cells with high expression level are screened by removing the pressure culture. Culturing the screened monoclonal cells to express the target antibody, centrifuging the culture solution, collecting supernatant, loading the supernatant onto a MabSelect SuRe LX column, eluting the target antibody protein by using an eluent with the pH of 3.0, neutralizing the target antibody protein to the pH of 7.0, and changing the purified antibody protein to PBS by using an ultrafiltration concentration tube.

Embodiment case 2: binding of bispecific antibodies to PD-L1 antigen

ELISA was used to detect binding of bispecific antibody to the antigen protein PD-L1. The purified antigen protein PD-L1-avi-his was expressed at a concentration of 0.5. Mu.g/ml and the ELISA plate was spread and left overnight at 4℃and the ELISA plate was washed 3 times with PBS+0.1% Tween20 solution and blocked with PBS-2.5% skim milk powder solution at 37℃for 1h. Washing 3 times with PBS+0.1% Tween20 solution, binding with bispecific antibody (first hole 1 μg/ml, 3-fold gradient dilution) to antigen, incubating with goat anti-human Fc-HRP secondary antibody, OPD development, 1M H ₂ SO ₄ And (5) terminating. The chromogenic ELISA plates were read with an microplate reader at OD490 and the readings were plotted against concentration (FIG. 2).

Binding of bispecific antibodies to PD-L1 antigen expressed on the surface of CHO cells was detected by flow cytometry (FACS). The concentration gradient bispecific antibody (first well 10. Mu.g/ml, 4-fold gradient dilution) was incubated with a CHO cell line expressing PD-L1 antigen (2E 5 cell/tube) and then detected with a second antibody with a fluorescent dye, sheep anti-human Fc-FITC. Cell binding of the detection antibodies was analyzed on a Beckman flow cytometer. Values were plotted against antibody concentration (fig. 4).

Embodiment 3: binding of bispecific antibodies to TIGIT antigens

ELISA was used to detect binding of bispecific antibody to antigen protein TIGIT, ELISA plates were plated at a concentration of 0.5. Mu.g/ml expressing purified antigen protein TIGIT-avi-his and left overnight at 4℃and washed 3 times with PBS+0.1% Tween20 solution and blocked with PBS-2.5% nonfat milk powder solution at 37℃for 1h. Washing 3 times with PBS+0.1% Tween20 solution, binding with bispecific antibody (first hole 1 μg/ml, 3-fold gradient dilution) to antigen, incubating with goat anti-human Fc-HRP secondary antibody, OPD development, 1M H ₂ SO ₄ And (5) terminating. The chromogenic ELISA plates were read with an microplate reader at OD490 and the readings were plotted against concentration (FIG. 3).

Binding of bispecific antibodies to TIGIT antigen expressed on CHO cell surface was detected by flow cytometry (FACS). The concentration gradient of bispecific antibody (first well 10. Mu.g/ml, 4-fold gradient dilution) was incubated with a CHO cell line expressing PD-L1 antigen (2E 5 cell/tube) and then detected with a second antibody against human Fc-FITC in sheep with a fluorescent dye. Cell binding of the detection antibodies was analyzed on a Beckman flow cytometer. Values were plotted against antibody concentration (fig. 5, fig. 6).

Embodiment 4: blocking Activity of bispecific antibodies against PD-L1/PD1

Blocking of the PD-L1 antigen expressed on the surface of CHO cells by bispecific antibodies was detected by flow cytometry (FACS). Gradient concentrations of bispecific antibody (first well 10. Mu.g/ml, 4-fold gradient dilution) and 1. Mu.g/ml of PD-1-mcFc antigen were incubated simultaneously with a CHO cell line expressing PD-L1 antigen (2E 5 cell/tube) and then detected with a second antibody of sheep anti-murine Fc-APC with fluorescent dye. The blocking activity of the detection antibodies was analyzed on a Beckman flow cytometer. Values were plotted against antibody concentration (fig. 7, 8).

Embodiment case 5: blocking Activity of bispecific antibodies against CD155/TIGIT

Blocking of TIGIT antigen expressed on CHO cell surface by bispecific antibodies was detected by flow cytometry (FACS). Gradient concentrations of bispecific antibody (first well 10. Mu.g/ml, 4-fold gradient dilution) and 1. Mu.g/ml of CD155-647 antigen were incubated simultaneously with the CHO cell line expressing TIGIT antigen (2E 5 cell/tube). The blocking activity of the detection antibodies was analyzed on a Beckman flow cytometer. Values were plotted against antibody concentration (fig. 9).

Embodiment 6: PD-1/PD-L1 and CD155/TIGIT pathway signaling

Bispecific antibodies were tested for their ability to simultaneously block the PD-1/PD-L1 and CD155/TIGIT pathways. We independently constructed Jurkat reporter cell lines and CHO-PD-L1-aAPC cell lines capable of expressing the receptor proteins PD-1, TIGIT and the luciferase NFAT reporter, to assess the biological activity of bispecific antibodies for simultaneous blocking of 2 signaling pathways. Specifically, CHO-PD-L1 cells (3E 4 cell/well) were plated overnight, and on the next day, concentration gradient bispecific antibody (406 nM in the first well, eight gradients diluted with 3-fold gradient), jurkat-PD1-TIGIT-NFAT-Luc cells (5E 4 cell/well) and 1. Mu.g/ml of final concentration CD155-mFc protein were incubated for 20min and then with the plated cells for another 4h. After addition of Bio-GloTM luciferases (from Promega) in the dark at room temperature for 10min, the assay was read on a plate reader (FLEX). Different types of bispecific antibodies and monoclonal control antibodies were shown to have different blocking activities on the PD-1/PD-L1 and TIGIT/CD155 signaling pathways (fig. 10).

Embodiment 7: bispecific antibody purity detection

The purity of the bispecific antibody was checked by means of size exclusion and, briefly, the purified bispecific antibody was checked on Waters' HPLC with TSK gelG3000SWXL molecular sieves. The purity of the bispecific antibodies one-step protein A formed by different structures and linker showed obvious differences (FIGS. 11, 12).

Embodiment case 8: PD-1/PD-L1/TIGIT three-humanized C57 mouse tumor model

The ability of bispecific antibodies to inhibit tumor growth in vivo was evaluated using a PD-1/PD-L1/TIGIT three humanized C57 mouse model. Briefly, PD-1/PD-L1/TIGIT trisonized C57 mice were purchased from Bai Chart Gene biotechnology Co. On the seventh day, animals were subcutaneously transplanted with the mouse colon cancer cell line MC38-PD-L1 (1E 6 cells/animal) that highly expressed the human PD-L1 antigen, and animal body weight and tumor size were examined. After the tumor volume average value is 40-80 mm3, grouping and starting IP administration, carrying out administration treatment on the bispecific antibody and the control antibody for 2 times a week, measuring the tumor volume and weighing the animal body weight for 2 times a week, and carrying out administration for four weeks. The bispecific antibodies tested were able to inhibit tumor growth better than the control single antibody, while tumor inhibition activity was no less than the control group of 2 single antibodies used in combination (fig. 13).

Sequence listing

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The principles and embodiments of the present application have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present application and its core ideas. The foregoing is merely illustrative of the preferred embodiments of this application, and it is noted that there is objectively no limit to the specific structure disclosed herein, since numerous modifications, adaptations and variations can be made by those skilled in the art without departing from the principles of the application, and the above-described features can be combined in any suitable manner; such modifications, variations and combinations, or the direct application of the inventive concepts and aspects to other applications without modification, are contemplated as falling within the scope of the present application.

SEQUENCE LISTING

<110> Anhui Annean bioengineering (group) Co., ltd

<120> bispecific antibodies against PD-L1 and TIGIT, their preparation and use

<130> 2020

<160> 38

<170> PatentIn version 3.5

<210> 1

<211> 2079

<212> DNA

<213> Artificial Sequence

<220>

<223> TPAmutC1 nucleotide

<400> 1

caggtgcagc tccaggagag cggccctgga ctggtgaagc cctctgagac cctcagcctg 60

acttgcacag tgagcggcgg atctatctct ggagcttaca tgtggagctg gatcaggcag 120

cctcccggca agtgcctgga atggattggg agtatcgtgc ctcacaggag cgccacctcc 180

tacaaccctt ctctgaaaag cagagtcaca atttctgtgg acacatccaa gaaccagttc 240

tctctgaaat tgtctagcgt taccgccgct gatacagcag tctactattg cgctaggtct 300

tacggaggca acagactgga ctattggggc cagggaacac tagtgacagt gagcagcggt 360

ggtggtggaa gtggtggagg tggatcagga ggtggaggaa gtgagctggt gctcacccag 420

agccctgcca cactgtccct ctctcctggc gaaagggcca cactgagctg tagagcttcc 480

cagggcgtgg gagaaaggct gtcttggtat caacagaagc ctggacaggc tcctagattg 540

ctgatctatt actctgccta ccgcgctact ggcgtgcctg ctcggttcag tggatctggc 600

agcggaacag acttcaccct gacaatcagc tccctggagc ctgaagactt tgccgtgtac 660

tattgccttc agaccttcca catccctttt acattcggct gcggaaccaa gctcgaaatc 720

aaggcttcca caaagggccc acaggtgcag cttgtggaga gcggaggagg cctggtgcag 780

cctggaggct ctctgagact ctcctgtacc gtgtctggca tcgacctgag ctcctatgac 840

atgacctggg tccgccaggc tccagggaag gggctggagt acatcggcta cattagctac 900

gtgtctcgga catactatgc cgacagcgtg aagggcagat tcaccatctc caaggacacc 960

tccaagaaca cggtgtatct gcagatgaac agcctgagag ccgaggacac ggccgtgtat 1020

tactgtgcca gagacaggcc cgatggcgct gccaccaacc tgtggggaca gggtacacta 1080

gtgacagtgt cctctgctag caccaagggc ccctccgtgt tccctctggc cccttcttcc 1140

aagtccacca gcggaggcac cgccgctctg ggctgcctcg tgaaagacta cttccccgag 1200

cccgtgaccg tgtcttggaa ctctggcgct ctgacctctg gcgtgcacac cttccctgct 1260

gtgctgcagt cctccggcct gtactctctg tcctccgtcg tgactgtgcc ctccagctct 1320

ctgggcaccc agacctacat ctgtaacgtg aaccacaagc cctccaacac caaggtggac 1380

aagaaggtgg aacctaagtc ttgtgacaaa acacacacct gccccccttg tcctgcccct 1440

gaactcctgg gcggaccctc tgtgtttctg ttccccccaa agcctaagga caccctgatg 1500

atctcccgga cccccgaagt gacctgcgtg gtggtggatg tgtcccacga agatcccgag 1560

gtgaagttca attggtacgt ggacggcgtg gaagtgcaca acgccaagac caagcctaga 1620

gaggaacagt acaactccac ctaccgggtg gtgtctgtgc tgaccgtgct gcaccaggac 1680

tggctgaacg gcaaagagta caagtgcaag gtgtccaaca aggctctgcc cgcccctatc 1740

gaaaagacca tcagcaaggc caagggccag ccccgggaac cccaggtgta cacactgcct 1800

ccaagcagag acgagctgac caagaatcag gtgtccctga cctgtctcgt gaagggcttc 1860

tacccctccg atatcgccgt ggaatgggag tccaacggcc agcctgagaa caactacaag 1920

accacccccc ctgtgctgga ctccgacggc tccttctttc tgtattctaa gctgaccgtg 1980

gacaagtccc ggtggcagca gggcaacgtg ttctcctgca gcgtgatgca cgaggccctg 2040

cacaaccact acacccagaa gtccctgtcc ctgtctccc 2079

<210> 2

<211> 693

<212> PRT

<213> Artificial Sequence

<220>

<223> TPAmutC1 amino acid

<400> 2

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Gly Ala

20 25 30

Tyr Met Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp

35 40 45

Ile Gly Ser Ile Val Pro His Arg Ser Ala Thr Ser Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Tyr Gly Gly Asn Arg Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Glu Leu Val Leu Thr Gln Ser Pro Ala Thr

130 135 140

Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

145 150 155 160

Gln Gly Val Gly Glu Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ala Pro Arg Leu Leu Ile Tyr Tyr Ser Ala Tyr Arg Ala Thr Gly Val

180 185 190

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln

210 215 220

Thr Phe His Ile Pro Phe Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys Ala Ser Thr Lys Gly Pro Gln Val Gln Leu Val Glu Ser Gly Gly

245 250 255

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Val Ser

260 265 270

Gly Ile Asp Leu Ser Ser Tyr Asp Met Thr Trp Val Arg Gln Ala Pro

275 280 285

Gly Lys Gly Leu Glu Tyr Ile Gly Tyr Ile Ser Tyr Val Ser Arg Thr

290 295 300

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Thr

305 310 315 320

Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

325 330 335

Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Asp Gly Ala Ala Thr

340 345 350

Asn Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

355 360 365

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

370 375 380

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

385 390 395 400

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

405 410 415

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

420 425 430

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

435 440 445

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

450 455 460

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

465 470 475 480

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

485 490 495

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

500 505 510

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

515 520 525

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

530 535 540

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

545 550 555 560

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

565 570 575

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

580 585 590

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

595 600 605

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

610 615 620

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

625 630 635 640

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

645 650 655

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

660 665 670

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

675 680 685

Leu Ser Leu Ser Pro

690

<210> 3

<211> 651

<212> DNA

<213> Artificial Sequence

<220>

<223> PD-L1 antibody light chain nucleotide

<400> 3

gacatccaga tgacacagag cccttccacc ctgtccgcca gcgtgggaga cagagtgacc 60

atcacttgcc agtccagcca gaacgtgtac tctaacaata gactgtcatg gtatcagcag 120

aagccaggga aggctcctaa gctcctgatc tattggacca gcttcctcgc ctccggagtg 180

ccatcaaggt tcagcggcag tggatctggg acagaattta ctctgaccat cagctccctg 240

cagcccgacg attttgccac ttattactgc gctggcggat acagcggcaa cctgtacacc 300

ttcggtcagg gtaccaagtt ggaaattaag cgtacggtgg ccgctccctc cgtgttcatc 360

ttcccacctt ccgacgagca gctgaagtcc ggcaccgctt ctgtcgtgtg cctgctgaac 420

aacttctacc cccgcgaggc caaggtgcag tggaaggtgg acaacgccct gcagtctggc 480

aactcccagg aatccgtgac cgagcaggac tccaaggaca gcacctactc cctgtcctcc 540

accctgaccc tgtccaaggc cgactacgag aagcacaagg tgtacgcctg cgaagtgacc 600

caccagggcc tgtctagccc cgtgaccaag tctttcaacc ggggagagtg c 651

<210> 4

<211> 217

<212> PRT

<213> Artificial Sequence

<220>

<223> PD-L1 antibody light chain amino acids

<400> 4

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Asn Val Tyr Ser Asn

20 25 30

Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly

85 90 95

Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

130 135 140

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155 160

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

180 185 190

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 5

<211> 2100

<212> DNA

<213> Artificial Sequence

<220>

<223> TPAmutC2 nucleotide

<400> 5

caggtgcagc tccaggagag cggccctgga ctggtgaagc cctctgagac cctcagcctg 60

acttgcacag tgagcggcgg atctatctct ggagcttaca tgtggagctg gatcaggcag 120

cctcccggca agtgcctgga atggattggg agtatcgtgc ctcacaggag cgccacctcc 180

tacaaccctt ctctgaaaag cagagtcaca atttctgtgg acacatccaa gaaccagttc 240

tctctgaaat tgtctagcgt taccgccgct gatacagcag tctactattg cgctaggtct 300

tacggaggca acagactgga ctattggggc cagggaacac tagtgacagt gagcagcggt 360

ggtggtggaa gtggtggagg tggatcagga ggtggaggaa gtgagctggt gctcacccag 420

agccctgcca cactgtccct ctctcctggc gaaagggcca cactgagctg tagagcttcc 480

cagggcgtgg gagaaaggct gtcttggtat caacagaagc ctggacaggc tcctagattg 540

ctgatctatt actctgccta ccgcgctact ggcgtgcctg ctcggttcag tggatctggc 600

agcggaacag acttcaccct gacaatcagc tccctggagc ctgaagactt tgccgtgtac 660

tattgccttc agaccttcca catccctttt acattcggct gcggaaccaa gctcgaaatc 720

aaggcttcca caaaaggacc atccgtcttc cctctggctc ctcaggtgca gcttgtggag 780

agcggaggag gcctggtgca gcctggaggc tctctgagac tctcctgtac cgtgtctggc 840

atcgacctga gctcctatga catgacctgg gtccgccagg ctccagggaa ggggctggag 900

tacatcggct acattagcta cgtgtctcgg acatactatg ccgacagcgt gaagggcaga 960

ttcaccatct ccaaggacac ctccaagaac acggtgtatc tgcagatgaa cagcctgaga 1020

gccgaggaca cggccgtgta ttactgtgcc agagacaggc ccgatggcgc tgccaccaac 1080

ctgtggggac agggtacact agtgacagtg tcctctgcta gcaccaaggg cccctccgtg 1140

ttccctctgg ccccttcttc caagtccacc agcggaggca ccgccgctct gggctgcctc 1200

gtgaaagact acttccccga gcccgtgacc gtgtcttgga actctggcgc tctgacctct 1260

ggcgtgcaca ccttccctgc tgtgctgcag tcctccggcc tgtactctct gtcctccgtc 1320

gtgactgtgc cctccagctc tctgggcacc cagacctaca tctgtaacgt gaaccacaag 1380

ccctccaaca ccaaggtgga caagaaggtg gaacctaagt cttgtgacaa aacacacacc 1440

tgcccccctt gtcctgcccc tgaactcctg ggcggaccct ctgtgtttct gttcccccca 1500

aagcctaagg acaccctgat gatctcccgg acccccgaag tgacctgcgt ggtggtggat 1560

gtgtcccacg aagatcccga ggtgaagttc aattggtacg tggacggcgt ggaagtgcac 1620

aacgccaaga ccaagcctag agaggaacag tacaactcca cctaccgggt ggtgtctgtg 1680

ctgaccgtgc tgcaccagga ctggctgaac ggcaaagagt acaagtgcaa ggtgtccaac 1740

aaggctctgc ccgcccctat cgaaaagacc atcagcaagg ccaagggcca gccccgggaa 1800

ccccaggtgt acacactgcc tccaagcaga gacgagctga ccaagaatca ggtgtccctg 1860

acctgtctcg tgaagggctt ctacccctcc gatatcgccg tggaatggga gtccaacggc 1920

cagcctgaga acaactacaa gaccaccccc cctgtgctgg actccgacgg ctccttcttt 1980

ctgtattcta agctgaccgt ggacaagtcc cggtggcagc agggcaacgt gttctcctgc 2040

agcgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgtc cctgtctccc 2100

<210> 6

<211> 700

<212> PRT

<213> Artificial Sequence

<220>

<223> TPAmutC2 amino acid

<400> 6

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Gly Ala

20 25 30

Tyr Met Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp

35 40 45

Ile Gly Ser Ile Val Pro His Arg Ser Ala Thr Ser Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Tyr Gly Gly Asn Arg Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Glu Leu Val Leu Thr Gln Ser Pro Ala Thr

130 135 140

Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

145 150 155 160

Gln Gly Val Gly Glu Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ala Pro Arg Leu Leu Ile Tyr Tyr Ser Ala Tyr Arg Ala Thr Gly Val

180 185 190

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln

210 215 220

Thr Phe His Ile Pro Phe Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Gln Val

245 250 255

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

260 265 270

Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr Asp Met

275 280 285

Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly Tyr

290 295 300

Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg

305 310 315 320

Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu Gln Met

325 330 335

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp

340 345 350

Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr Leu Val

355 360 365

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

370 375 380

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

385 390 395 400

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

405 410 415

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

420 425 430

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

435 440 445

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

450 455 460

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

465 470 475 480

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

485 490 495

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

500 505 510

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

515 520 525

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

530 535 540

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

545 550 555 560

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

565 570 575

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

580 585 590

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

595 600 605

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

610 615 620

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

625 630 635 640

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

645 650 655

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

660 665 670

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

675 680 685

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

690 695 700

<210> 7

<211> 2076

<212> DNA

<213> Artificial Sequence

<220>

<223> TPAmutC3 nucleotide

<400> 7

caggtgcagc tccaggagag cggccctgga ctggtgaagc cctctgagac cctcagcctg 60

acttgcacag tgagcggcgg atctatctct ggagcttaca tgtggagctg gatcaggcag 120

cctcccggca agtgcctgga atggattggg agtatcgtgc ctcacaggag cgccacctcc 180

tacaaccctt ctctgaaaag cagagtcaca atttctgtgg acacatccaa gaaccagttc 240

tctctgaaat tgtctagcgt taccgccgct gatacagcag tctactattg cgctaggtct 300

tacggaggca acagactgga ctattggggc cagggaacac tagtgacagt gagcagcggt 360

ggtggtggaa gtggtggagg tggatcagga ggtggaggaa gtgagctggt gctcacccag 420

agccctgcca cactgtccct ctctcctggc gaaagggcca cactgagctg tagagcttcc 480

cagggcgtgg gagaaaggct gtcttggtat caacagaagc ctggacaggc tcctagattg 540

ctgatctatt actctgccta ccgcgctact ggcgtgcctg ctcggttcag tggatctggc 600

agcggaacag acttcaccct gacaatcagc tccctggagc ctgaagactt tgccgtgtac 660

tattgccttc agaccttcca catccctttt acattcggct gcggaaccaa gctcgaaatc 720

aagaccgtgg ccgcccctca ggtgcagctt gtggagagcg gaggaggcct ggtgcagcct 780

ggaggctctc tgagactctc ctgtaccgtg tctggcatcg acctgagctc ctatgacatg 840

acctgggtcc gccaggctcc agggaagggg ctggagtaca tcggctacat tagctacgtg 900

tctcggacat actatgccga cagcgtgaag ggcagattca ccatctccaa ggacacctcc 960

aagaacacgg tgtatctgca gatgaacagc ctgagagccg aggacacggc cgtgtattac 1020

tgtgccagag acaggcccga tggcgctgcc accaacctgt ggggacaggg tacactagtg 1080

acagtgtcct ctgctagcac caagggcccc tccgtgttcc ctctggcccc ttcttccaag 1140

tccaccagcg gaggcaccgc cgctctgggc tgcctcgtga aagactactt ccccgagccc 1200

gtgaccgtgt cttggaactc tggcgctctg acctctggcg tgcacacctt ccctgctgtg 1260

ctgcagtcct ccggcctgta ctctctgtcc tccgtcgtga ctgtgccctc cagctctctg 1320

ggcacccaga cctacatctg taacgtgaac cacaagccct ccaacaccaa ggtggacaag 1380

aaggtggaac ctaagtcttg tgacaaaaca cacacctgcc ccccttgtcc tgcccctgaa 1440

ctcctgggcg gaccctctgt gtttctgttc cccccaaagc ctaaggacac cctgatgatc 1500

tcccggaccc ccgaagtgac ctgcgtggtg gtggatgtgt cccacgaaga tcccgaggtg 1560

aagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcctagagag 1620

gaacagtaca actccaccta ccgggtggtg tctgtgctga ccgtgctgca ccaggactgg 1680

ctgaacggca aagagtacaa gtgcaaggtg tccaacaagg ctctgcccgc ccctatcgaa 1740

aagaccatca gcaaggccaa gggccagccc cgggaacccc aggtgtacac actgcctcca 1800

agcagagacg agctgaccaa gaatcaggtg tccctgacct gtctcgtgaa gggcttctac 1860

ccctccgata tcgccgtgga atgggagtcc aacggccagc ctgagaacaa ctacaagacc 1920

accccccctg tgctggactc cgacggctcc ttctttctgt attctaagct gaccgtggac 1980

aagtcccggt ggcagcaggg caacgtgttc tcctgcagcg tgatgcacga ggccctgcac 2040

aaccactaca cccagaagtc cctgtccctg tctccc 2076

<210> 8

<211> 692

<212> PRT

<213> Artificial Sequence

<220>

<223> TPAmutC3 amino acid

<400> 8

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Gly Ala

20 25 30

Tyr Met Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp

35 40 45

Ile Gly Ser Ile Val Pro His Arg Ser Ala Thr Ser Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Tyr Gly Gly Asn Arg Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Glu Leu Val Leu Thr Gln Ser Pro Ala Thr

130 135 140

Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

145 150 155 160

Gln Gly Val Gly Glu Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ala Pro Arg Leu Leu Ile Tyr Tyr Ser Ala Tyr Arg Ala Thr Gly Val

180 185 190

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln

210 215 220

Thr Phe His Ile Pro Phe Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys Thr Val Ala Ala Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly

245 250 255

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Val Ser Gly

260 265 270

Ile Asp Leu Ser Ser Tyr Asp Met Thr Trp Val Arg Gln Ala Pro Gly

275 280 285

Lys Gly Leu Glu Tyr Ile Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr

290 295 300

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser

305 310 315 320

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

325 330 335

Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn

340 345 350

Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

355 360 365

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

370 375 380

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

385 390 395 400

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

405 410 415

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

420 425 430

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

435 440 445

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

450 455 460

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

465 470 475 480

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

485 490 495

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

500 505 510

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

515 520 525

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

530 535 540

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

545 550 555 560

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

565 570 575

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

580 585 590

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

595 600 605

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

610 615 620

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

625 630 635 640

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

645 650 655

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

660 665 670

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

675 680 685

Ser Leu Ser Pro

690

<210> 9

<211> 2097

<212> DNA

<213> Artificial Sequence

<220>

<223> TPAmutC4 nucleotide

<400> 9

caggtgcagc tccaggagag cggccctgga ctggtgaagc cctctgagac cctcagcctg 60

acttgcacag tgagcggcgg atctatctct ggagcttaca tgtggagctg gatcaggcag 120

cctcccggca agtgcctgga atggattggg agtatcgtgc ctcacaggag cgccacctcc 180

tacaaccctt ctctgaaaag cagagtcaca atttctgtgg acacatccaa gaaccagttc 240

tctctgaaat tgtctagcgt taccgccgct gatacagcag tctactattg cgctaggtct 300

tacggaggca acagactgga ctattggggc cagggaacac tagtgacagt gagcagcggt 360

ggtggtggaa gtggtggagg tggatcagga ggtggaggaa gtgagctggt gctcacccag 420

agccctgcca cactgtccct ctctcctggc gaaagggcca cactgagctg tagagcttcc 480

cagggcgtgg gagaaaggct gtcttggtat caacagaagc ctggacaggc tcctagattg 540

ctgatctatt actctgccta ccgcgctact ggcgtgcctg ctcggttcag tggatctggc 600

agcggaacag acttcaccct gacaatcagc tccctggagc ctgaagactt tgccgtgtac 660

tattgccttc agaccttcca catccctttt acattcggct gcggaaccaa gctcgaaatc 720

aagaccgtgg ccgccccttc cgtgttcatc ttccctcccc aggtgcagct tgtggagagc 780

ggaggaggcc tggtgcagcc tggaggctct ctgagactct cctgtaccgt gtctggcatc 840

gacctgagct cctatgacat gacctgggtc cgccaggctc cagggaaggg gctggagtac 900

atcggctaca ttagctacgt gtctcggaca tactatgccg acagcgtgaa gggcagattc 960

accatctcca aggacacctc caagaacacg gtgtatctgc agatgaacag cctgagagcc 1020

gaggacacgg ccgtgtatta ctgtgccaga gacaggcccg atggcgctgc caccaacctg 1080

tggggacagg gtacactagt gacagtgtcc tctgctagca ccaagggccc ctccgtgttc 1140

cctctggccc cttcttccaa gtccaccagc ggaggcaccg ccgctctggg ctgcctcgtg 1200

aaagactact tccccgagcc cgtgaccgtg tcttggaact ctggcgctct gacctctggc 1260

gtgcacacct tccctgctgt gctgcagtcc tccggcctgt actctctgtc ctccgtcgtg 1320

actgtgccct ccagctctct gggcacccag acctacatct gtaacgtgaa ccacaagccc 1380

tccaacacca aggtggacaa gaaggtggaa cctaagtctt gtgacaaaac acacacctgc 1440

cccccttgtc ctgcccctga actcctgggc ggaccctctg tgtttctgtt ccccccaaag 1500

cctaaggaca ccctgatgat ctcccggacc cccgaagtga cctgcgtggt ggtggatgtg 1560

tcccacgaag atcccgaggt gaagttcaat tggtacgtgg acggcgtgga agtgcacaac 1620

gccaagacca agcctagaga ggaacagtac aactccacct accgggtggt gtctgtgctg 1680

accgtgctgc accaggactg gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 1740

gctctgcccg cccctatcga aaagaccatc agcaaggcca agggccagcc ccgggaaccc 1800

caggtgtaca cactgcctcc aagcagagac gagctgacca agaatcaggt gtccctgacc 1860

tgtctcgtga agggcttcta cccctccgat atcgccgtgg aatgggagtc caacggccag 1920

cctgagaaca actacaagac caccccccct gtgctggact ccgacggctc cttctttctg 1980

tattctaagc tgaccgtgga caagtcccgg tggcagcagg gcaacgtgtt ctcctgcagc 2040

gtgatgcacg aggccctgca caaccactac acccagaagt ccctgtccct gtctccc 2097

<210> 10

<211> 699

<212> PRT

<213> Artificial Sequence

<220>

<223> TPAmutC4 amino acid

<400> 10

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Gly Ala

20 25 30

Tyr Met Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp

35 40 45

Ile Gly Ser Ile Val Pro His Arg Ser Ala Thr Ser Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Tyr Gly Gly Asn Arg Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Glu Leu Val Leu Thr Gln Ser Pro Ala Thr

130 135 140

Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

145 150 155 160

Gln Gly Val Gly Glu Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ala Pro Arg Leu Leu Ile Tyr Tyr Ser Ala Tyr Arg Ala Thr Gly Val

180 185 190

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln

210 215 220

Thr Phe His Ile Pro Phe Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Gln Val Gln

245 250 255

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

260 265 270

Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr Asp Met Thr

275 280 285

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly Tyr Ile

290 295 300

Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

305 310 315 320

Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu Gln Met Asn

325 330 335

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg

340 345 350

Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr Leu Val Thr

355 360 365

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

370 375 380

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

385 390 395 400

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

405 410 415

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

420 425 430

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

435 440 445

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

450 455 460

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

465 470 475 480

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

485 490 495

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

500 505 510

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

515 520 525

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

530 535 540

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

545 550 555 560

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

565 570 575

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

580 585 590

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

595 600 605

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

610 615 620

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

625 630 635 640

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

645 650 655

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

660 665 670

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

675 680 685

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

690 695

<210> 11

<211> 2094

<212> DNA

<213> Artificial Sequence

<220>

<223> TPA5mutC1 nucleotide

<400> 11

caggtgcagc tccaggagag cggccctgga ctggtgaagc cctctgagac cctcagcctg 60

acttgcacag tgagcggcgg atctatctct ggagcttaca tgtggagctg gatcaggcag 120

cctcccggca agtgcctgga atggattggg agtatcgtgc ctcacaggag cgccacctcc 180

tacaaccctt ctctgaaaag cagagtcaca atttctgtgg acacatccaa gaaccagttc 240

tctctgaaat tgtctagcgt taccgccgct gatacagcag tctactattg cgctaggtct 300

tacggaggca acagactgga ctattggggc cagggaacac tagtgacagt gagcagcggt 360

ggtggtggaa gtggtggagg tggatcagga ggtggaggaa gtggaggagg aggttcagag 420

ctggtgctca cccagagccc tgccacactg tccctctctc ctggcgaaag ggccacactg 480

agctgtagag cttcccaggg cgtgggagaa aggctgtctt ggtatcaaca gaagcctgga 540

caggctccta gattgctgat ctattactct gcctaccgcg ctactggcgt gcctgctcgg 600

ttcagtggat ctggcagcgg aacagacttc accctgacaa tcagctccct ggagcctgaa 660

gactttgccg tgtactattg ccttcagacc ttccacatcc cttttacatt cggctgcgga 720

accaagctcg aaatcaaggc ttccacaaag ggcccacagg tgcagcttgt ggagagcgga 780

ggaggcctgg tgcagcctgg aggctctctg agactctcct gtaccgtgtc tggcatcgac 840

ctgagctcct atgacatgac ctgggtccgc caggctccag ggaaggggct ggagtacatc 900

ggctacatta gctacgtgtc tcggacatac tatgccgaca gcgtgaaggg cagattcacc 960

atctccaagg acacctccaa gaacacggtg tatctgcaga tgaacagcct gagagccgag 1020

gacacggccg tgtattactg tgccagagac aggcccgatg gcgctgccac caacctgtgg 1080

ggacagggta cactagtgac agtgtcctct gctagcacca agggcccctc cgtgttccct 1140

ctggcccctt cttccaagtc caccagcgga ggcaccgccg ctctgggctg cctcgtgaaa 1200

gactacttcc ccgagcccgt gaccgtgtct tggaactctg gcgctctgac ctctggcgtg 1260

cacaccttcc ctgctgtgct gcagtcctcc ggcctgtact ctctgtcctc cgtcgtgact 1320

gtgccctcca gctctctggg cacccagacc tacatctgta acgtgaacca caagccctcc 1380

aacaccaagg tggacaagaa ggtggaacct aagtcttgtg acaaaacaca cacctgcccc 1440

ccttgtcctg cccctgaact cctgggcgga ccctctgtgt ttctgttccc cccaaagcct 1500

aaggacaccc tgatgatctc ccggaccccc gaagtgacct gcgtggtggt ggatgtgtcc 1560

cacgaagatc ccgaggtgaa gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc 1620

aagaccaagc ctagagagga acagtacaac tccacctacc gggtggtgtc tgtgctgacc 1680

gtgctgcacc aggactggct gaacggcaaa gagtacaagt gcaaggtgtc caacaaggct 1740

ctgcccgccc ctatcgaaaa gaccatcagc aaggccaagg gccagccccg ggaaccccag 1800

gtgtacacac tgcctccaag cagagacgag ctgaccaaga atcaggtgtc cctgacctgt 1860

ctcgtgaagg gcttctaccc ctccgatatc gccgtggaat gggagtccaa cggccagcct 1920

gagaacaact acaagaccac cccccctgtg ctggactccg acggctcctt ctttctgtat 1980

tctaagctga ccgtggacaa gtcccggtgg cagcagggca acgtgttctc ctgcagcgtg 2040

atgcacgagg ccctgcacaa ccactacacc cagaagtccc tgtccctgtc tccc 2094

<210> 12

<211> 698

<212> PRT

<213> Artificial Sequence

<220>

<223> TPA5mutC1 amino acids

<400> 12

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Gly Ala

20 25 30

Tyr Met Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp

35 40 45

Ile Gly Ser Ile Val Pro His Arg Ser Ala Thr Ser Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Tyr Gly Gly Asn Arg Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Leu Thr

130 135 140

Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

145 150 155 160

Ser Cys Arg Ala Ser Gln Gly Val Gly Glu Arg Leu Ser Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Tyr Ser Ala Tyr

180 185 190

Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val

210 215 220

Tyr Tyr Cys Leu Gln Thr Phe His Ile Pro Phe Thr Phe Gly Cys Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Ala Ser Thr Lys Gly Pro Gln Val Gln Leu

245 250 255

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

260 265 270

Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr Asp Met Thr Trp

275 280 285

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly Tyr Ile Ser

290 295 300

Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

305 310 315 320

Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser

325 330 335

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro

340 345 350

Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr Leu Val Thr Val

355 360 365

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

370 375 380

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

385 390 395 400

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

405 410 415

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

420 425 430

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

435 440 445

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

450 455 460

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

465 470 475 480

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

485 490 495

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

500 505 510

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

515 520 525

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

530 535 540

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

545 550 555 560

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

565 570 575

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

580 585 590

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

595 600 605

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

610 615 620

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

625 630 635 640

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

645 650 655

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

660 665 670

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

675 680 685

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

690 695

<210> 13

<211> 2115

<212> DNA

<213> Artificial Sequence

<220>

<223> TPA5mutC2 nucleotide

<400> 13

caggtgcagc tccaggagag cggccctgga ctggtgaagc cctctgagac cctcagcctg 60

acttgcacag tgagcggcgg atctatctct ggagcttaca tgtggagctg gatcaggcag 120

cctcccggca agtgcctgga atggattggg agtatcgtgc ctcacaggag cgccacctcc 180

tacaaccctt ctctgaaaag cagagtcaca atttctgtgg acacatccaa gaaccagttc 240

tctctgaaat tgtctagcgt taccgccgct gatacagcag tctactattg cgctaggtct 300

tacggaggca acagactgga ctattggggc cagggaacac tagtgacagt gagcagcggt 360

ggtggtggaa gtggtggagg tggatcagga ggtggaggaa gtggaggagg aggttcagag 420

ctggtgctca cccagagccc tgccacactg tccctctctc ctggcgaaag ggccacactg 480

agctgtagag cttcccaggg cgtgggagaa aggctgtctt ggtatcaaca gaagcctgga 540

caggctccta gattgctgat ctattactct gcctaccgcg ctactggcgt gcctgctcgg 600

ttcagtggat ctggcagcgg aacagacttc accctgacaa tcagctccct ggagcctgaa 660

gactttgccg tgtactattg ccttcagacc ttccacatcc cttttacatt cggctgcgga 720

accaagctcg aaatcaaggc ttccacaaaa ggaccatccg tcttccctct ggctcctcag 780

gtgcagcttg tggagagcgg aggaggcctg gtgcagcctg gaggctctct gagactctcc 840

tgtaccgtgt ctggcatcga cctgagctcc tatgacatga cctgggtccg ccaggctcca 900

gggaaggggc tggagtacat cggctacatt agctacgtgt ctcggacata ctatgccgac 960

agcgtgaagg gcagattcac catctccaag gacacctcca agaacacggt gtatctgcag 1020

atgaacagcc tgagagccga ggacacggcc gtgtattact gtgccagaga caggcccgat 1080

ggcgctgcca ccaacctgtg gggacagggt acactagtga cagtgtcctc tgctagcacc 1140

aagggcccct ccgtgttccc tctggcccct tcttccaagt ccaccagcgg aggcaccgcc 1200

gctctgggct gcctcgtgaa agactacttc cccgagcccg tgaccgtgtc ttggaactct 1260

ggcgctctga cctctggcgt gcacaccttc cctgctgtgc tgcagtcctc cggcctgtac 1320

tctctgtcct ccgtcgtgac tgtgccctcc agctctctgg gcacccagac ctacatctgt 1380

aacgtgaacc acaagccctc caacaccaag gtggacaaga aggtggaacc taagtcttgt 1440

gacaaaacac acacctgccc cccttgtcct gcccctgaac tcctgggcgg accctctgtg 1500

tttctgttcc ccccaaagcc taaggacacc ctgatgatct cccggacccc cgaagtgacc 1560

tgcgtggtgg tggatgtgtc ccacgaagat cccgaggtga agttcaattg gtacgtggac 1620

ggcgtggaag tgcacaacgc caagaccaag cctagagagg aacagtacaa ctccacctac 1680

cgggtggtgt ctgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag 1740

tgcaaggtgt ccaacaaggc tctgcccgcc cctatcgaaa agaccatcag caaggccaag 1800

ggccagcccc gggaacccca ggtgtacaca ctgcctccaa gcagagacga gctgaccaag 1860

aatcaggtgt ccctgacctg tctcgtgaag ggcttctacc cctccgatat cgccgtggaa 1920

tgggagtcca acggccagcc tgagaacaac tacaagacca ccccccctgt gctggactcc 1980

gacggctcct tctttctgta ttctaagctg accgtggaca agtcccggtg gcagcagggc 2040

aacgtgttct cctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 2100

ctgtccctgt ctccc 2115

<210> 14

<211> 705

<212> PRT

<213> Artificial Sequence

<220>

<223> TPA5mutC2 amino acids

<400> 14

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Gly Ala

20 25 30

Tyr Met Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp

35 40 45

Ile Gly Ser Ile Val Pro His Arg Ser Ala Thr Ser Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Tyr Gly Gly Asn Arg Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Leu Thr

130 135 140

Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

145 150 155 160

Ser Cys Arg Ala Ser Gln Gly Val Gly Glu Arg Leu Ser Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Tyr Ser Ala Tyr

180 185 190

Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val

210 215 220

Tyr Tyr Cys Leu Gln Thr Phe His Ile Pro Phe Thr Phe Gly Cys Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

245 250 255

Leu Ala Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

260 265 270

Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu

275 280 285

Ser Ser Tyr Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

290 295 300

Glu Tyr Ile Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp

305 310 315 320

Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr

325 330 335

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

340 345 350

Tyr Cys Ala Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly

355 360 365

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

370 375 380

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

385 390 395 400

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

405 410 415

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

420 425 430

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

435 440 445

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

450 455 460

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

465 470 475 480

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

485 490 495

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

500 505 510

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

515 520 525

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

530 535 540

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

545 550 555 560

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

565 570 575

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

580 585 590

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

595 600 605

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

610 615 620

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

625 630 635 640

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

645 650 655

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

660 665 670

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

675 680 685

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

690 695 700

Pro

705

<210> 15

<211> 2091

<212> DNA

<213> Artificial Sequence

<220>

<223> TPA5mutC3 nucleotide

<400> 15

caggtgcagc tccaggagag cggccctgga ctggtgaagc cctctgagac cctcagcctg 60

acttgcacag tgagcggcgg atctatctct ggagcttaca tgtggagctg gatcaggcag 120

cctcccggca agtgcctgga atggattggg agtatcgtgc ctcacaggag cgccacctcc 180

tacaaccctt ctctgaaaag cagagtcaca atttctgtgg acacatccaa gaaccagttc 240

tctctgaaat tgtctagcgt taccgccgct gatacagcag tctactattg cgctaggtct 300

tacggaggca acagactgga ctattggggc cagggaacac tagtgacagt gagcagcggt 360

ggtggtggaa gtggtggagg tggatcagga ggtggaggaa gtggaggagg aggttcagag 420

ctggtgctca cccagagccc tgccacactg tccctctctc ctggcgaaag ggccacactg 480

agctgtagag cttcccaggg cgtgggagaa aggctgtctt ggtatcaaca gaagcctgga 540

caggctccta gattgctgat ctattactct gcctaccgcg ctactggcgt gcctgctcgg 600

ttcagtggat ctggcagcgg aacagacttc accctgacaa tcagctccct ggagcctgaa 660

gactttgccg tgtactattg ccttcagacc ttccacatcc cttttacatt cggctgcgga 720

accaagctcg aaatcaagac cgtggccgcc cctcaggtgc agcttgtgga gagcggagga 780

ggcctggtgc agcctggagg ctctctgaga ctctcctgta ccgtgtctgg catcgacctg 840

agctcctatg acatgacctg ggtccgccag gctccaggga aggggctgga gtacatcggc 900

tacattagct acgtgtctcg gacatactat gccgacagcg tgaagggcag attcaccatc 960

tccaaggaca cctccaagaa cacggtgtat ctgcagatga acagcctgag agccgaggac 1020

acggccgtgt attactgtgc cagagacagg cccgatggcg ctgccaccaa cctgtgggga 1080

cagggtacac tagtgacagt gtcctctgct agcaccaagg gcccctccgt gttccctctg 1140

gccccttctt ccaagtccac cagcggaggc accgccgctc tgggctgcct cgtgaaagac 1200

tacttccccg agcccgtgac cgtgtcttgg aactctggcg ctctgacctc tggcgtgcac 1260

accttccctg ctgtgctgca gtcctccggc ctgtactctc tgtcctccgt cgtgactgtg 1320

ccctccagct ctctgggcac ccagacctac atctgtaacg tgaaccacaa gccctccaac 1380

accaaggtgg acaagaaggt ggaacctaag tcttgtgaca aaacacacac ctgcccccct 1440

tgtcctgccc ctgaactcct gggcggaccc tctgtgtttc tgttcccccc aaagcctaag 1500

gacaccctga tgatctcccg gacccccgaa gtgacctgcg tggtggtgga tgtgtcccac 1560

gaagatcccg aggtgaagtt caattggtac gtggacggcg tggaagtgca caacgccaag 1620

accaagccta gagaggaaca gtacaactcc acctaccggg tggtgtctgt gctgaccgtg 1680

ctgcaccagg actggctgaa cggcaaagag tacaagtgca aggtgtccaa caaggctctg 1740

cccgccccta tcgaaaagac catcagcaag gccaagggcc agccccggga accccaggtg 1800

tacacactgc ctccaagcag agacgagctg accaagaatc aggtgtccct gacctgtctc 1860

gtgaagggct tctacccctc cgatatcgcc gtggaatggg agtccaacgg ccagcctgag 1920

aacaactaca agaccacccc ccctgtgctg gactccgacg gctccttctt tctgtattct 1980

aagctgaccg tggacaagtc ccggtggcag cagggcaacg tgttctcctg cagcgtgatg 2040

cacgaggccc tgcacaacca ctacacccag aagtccctgt ccctgtctcc c 2091

<210> 16

<211> 697

<212> PRT

<213> Artificial Sequence

<220>

<223> TPA5mutC3 amino acids

<400> 16

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Gly Ala

20 25 30

Tyr Met Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp

35 40 45

Ile Gly Ser Ile Val Pro His Arg Ser Ala Thr Ser Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Tyr Gly Gly Asn Arg Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Leu Thr

130 135 140

Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

145 150 155 160

Ser Cys Arg Ala Ser Gln Gly Val Gly Glu Arg Leu Ser Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Tyr Ser Ala Tyr

180 185 190

Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val

210 215 220

Tyr Tyr Cys Leu Gln Thr Phe His Ile Pro Phe Thr Phe Gly Cys Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Thr Val Ala Ala Pro Gln Val Gln Leu Val

245 250 255

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

260 265 270

Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr Asp Met Thr Trp Val

275 280 285

Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly Tyr Ile Ser Tyr

290 295 300

Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

305 310 315 320

Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu

325 330 335

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Asp

340 345 350

Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser

355 360 365

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

370 375 380

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

385 390 395 400

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

405 410 415

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

420 425 430

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

435 440 445

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

450 455 460

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

465 470 475 480

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

485 490 495

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

500 505 510

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

515 520 525

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

530 535 540

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

545 550 555 560

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

565 570 575

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

580 585 590

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

595 600 605

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

610 615 620

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

625 630 635 640

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

645 650 655

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

660 665 670

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

675 680 685

Thr Gln Lys Ser Leu Ser Leu Ser Pro

690 695

<210> 17

<211> 2112

<212> DNA

<213> Artificial Sequence

<220>

<223> TPA5mutC4 nucleotide

<400> 17

caggtgcagc tccaggagag cggccctgga ctggtgaagc cctctgagac cctcagcctg 60

acttgcacag tgagcggcgg atctatctct ggagcttaca tgtggagctg gatcaggcag 120

cctcccggca agtgcctgga atggattggg agtatcgtgc ctcacaggag cgccacctcc 180

tacaaccctt ctctgaaaag cagagtcaca atttctgtgg acacatccaa gaaccagttc 240

tctctgaaat tgtctagcgt taccgccgct gatacagcag tctactattg cgctaggtct 300

tacggaggca acagactgga ctattggggc cagggaacac tagtgacagt gagcagcggt 360

ggtggtggaa gtggtggagg tggatcagga ggtggaggaa gtggaggagg aggttcagag 420

ctggtgctca cccagagccc tgccacactg tccctctctc ctggcgaaag ggccacactg 480

agctgtagag cttcccaggg cgtgggagaa aggctgtctt ggtatcaaca gaagcctgga 540

caggctccta gattgctgat ctattactct gcctaccgcg ctactggcgt gcctgctcgg 600

ttcagtggat ctggcagcgg aacagacttc accctgacaa tcagctccct ggagcctgaa 660

gactttgccg tgtactattg ccttcagacc ttccacatcc cttttacatt cggctgcgga 720

accaagctcg aaatcaagac cgtggccgcc ccttccgtgt tcatcttccc tccccaggtg 780

cagcttgtgg agagcggagg aggcctggtg cagcctggag gctctctgag actctcctgt 840

accgtgtctg gcatcgacct gagctcctat gacatgacct gggtccgcca ggctccaggg 900

aaggggctgg agtacatcgg ctacattagc tacgtgtctc ggacatacta tgccgacagc 960

gtgaagggca gattcaccat ctccaaggac acctccaaga acacggtgta tctgcagatg 1020

aacagcctga gagccgagga cacggccgtg tattactgtg ccagagacag gcccgatggc 1080

gctgccacca acctgtgggg acagggtaca ctagtgacag tgtcctctgc tagcaccaag 1140

ggcccctccg tgttccctct ggccccttct tccaagtcca ccagcggagg caccgccgct 1200

ctgggctgcc tcgtgaaaga ctacttcccc gagcccgtga ccgtgtcttg gaactctggc 1260

gctctgacct ctggcgtgca caccttccct gctgtgctgc agtcctccgg cctgtactct 1320

ctgtcctccg tcgtgactgt gccctccagc tctctgggca cccagaccta catctgtaac 1380

gtgaaccaca agccctccaa caccaaggtg gacaagaagg tggaacctaa gtcttgtgac 1440

aaaacacaca cctgcccccc ttgtcctgcc cctgaactcc tgggcggacc ctctgtgttt 1500

ctgttccccc caaagcctaa ggacaccctg atgatctccc ggacccccga agtgacctgc 1560

gtggtggtgg atgtgtccca cgaagatccc gaggtgaagt tcaattggta cgtggacggc 1620

gtggaagtgc acaacgccaa gaccaagcct agagaggaac agtacaactc cacctaccgg 1680

gtggtgtctg tgctgaccgt gctgcaccag gactggctga acggcaaaga gtacaagtgc 1740

aaggtgtcca acaaggctct gcccgcccct atcgaaaaga ccatcagcaa ggccaagggc 1800

cagccccggg aaccccaggt gtacacactg cctccaagca gagacgagct gaccaagaat 1860

caggtgtccc tgacctgtct cgtgaagggc ttctacccct ccgatatcgc cgtggaatgg 1920

gagtccaacg gccagcctga gaacaactac aagaccaccc cccctgtgct ggactccgac 1980

ggctccttct ttctgtattc taagctgacc gtggacaagt cccggtggca gcagggcaac 2040

gtgttctcct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 2100

tccctgtctc cc 2112

<210> 18

<211> 704

<212> PRT

<213> Artificial Sequence

<220>

<223> TPA5mutC4 amino acids

<400> 18

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Gly Ala

20 25 30

Tyr Met Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp

35 40 45

Ile Gly Ser Ile Val Pro His Arg Ser Ala Thr Ser Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Tyr Gly Gly Asn Arg Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Leu Thr

130 135 140

Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

145 150 155 160

Ser Cys Arg Ala Ser Gln Gly Val Gly Glu Arg Leu Ser Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Tyr Ser Ala Tyr

180 185 190

Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val

210 215 220

Tyr Tyr Cys Leu Gln Thr Phe His Ile Pro Phe Thr Phe Gly Cys Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Thr Val Ala Ala Pro Ser Val Phe Ile Phe

245 250 255

Pro Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

260 265 270

Gly Gly Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser

275 280 285

Ser Tyr Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

290 295 300

Tyr Ile Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser

305 310 315 320

Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val

325 330 335

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

340 345 350

Cys Ala Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln

355 360 365

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

370 375 380

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

385 390 395 400

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

405 410 415

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

420 425 430

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

435 440 445

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

450 455 460

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

465 470 475 480

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

485 490 495

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

500 505 510

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

515 520 525

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

530 535 540

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

545 550 555 560

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

565 570 575

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

580 585 590

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

595 600 605

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

610 615 620

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

625 630 635 640

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

645 650 655

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

660 665 670

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

675 680 685

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

690 695 700

<210> 19

<211> 2097

<212> DNA

<213> Artificial Sequence

<220>

<223> TPC1 nucleotide

<400> 19

caggtgcagc ttgtggagag cggaggaggc ctggtgcagc ctggaggctc tctgagactc 60

tcctgtaccg tgtctggcat cgacctgagc tcctatgaca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagta catcggctac attagctacg tgtctcggac atactatgcc 180

gacagcgtga agggcagatt caccatctcc aaggacacct ccaagaacac ggtgtatctg 240

cagatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgccag agacaggccc 300

gatggcgctg ccaccaacct gtggggacag ggtacactag tgacagtgtc ctctgctagc 360

accaagggcc cctccgtgtt ccctctggcc ccttcttcca agtccaccag cggaggcacc 420

gccgctctgg gctgcctcgt gaaagactac ttccccgagc ccgtgaccgt gtcttggaac 480

tctggcgctc tgacctctgg cgtgcacacc ttccctgctg tgctgcagtc ctccggcctg 540

tactctctgt cctccgtcgt gactgtgccc tccagctctc tgggcaccca gacctacatc 600

tgtaacgtga accacaagcc ctccaacacc aaggtggaca agaaggtgga acctaagtct 660

tgtgacaaaa cacacacctg ccccccttgt cctgcccctg aactcctggg cggaccctct 720

gtgtttctgt tccccccaaa gcctaaggac accctgatga tctcccggac ccccgaagtg 780

acctgcgtgg tggtggatgt gtcccacgaa gatcccgagg tgaagttcaa ttggtacgtg 840

gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc 900

taccgggtgg tgtctgtgct gaccgtgctg caccaggact ggctgaacgg caaagagtac 960

aagtgcaagg tgtccaacaa ggctctgccc gcccctatcg aaaagaccat cagcaaggcc 1020

aagggccagc cccgggaacc ccaggtgtac acactgcctc caagcagaga cgagctgacc 1080

aagaatcagg tgtccctgac ctgtctcgtg aagggcttct acccctccga tatcgccgtg 1140

gaatgggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1200

tccgacggct ccttctttct gtattctaag ctgaccgtgg acaagtcccg gtggcagcag 1260

ggcaacgtgt tctcctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag 1320

tccctgtccc tgtctcccgg aaagggtggt ggaggatcag gtggtggagg aagtcaggtg 1380

cagctccagg agagcggccc tggactggtg aagccctctg agaccctcag cctgacttgc 1440

acagtgagcg gcggatctat ctctggagct tacatgtgga gctggatcag gcagcctccc 1500

ggcaagggac tggaatggat tgggagtatc gtgcctcaca ggagcgccac ctcctacaac 1560

ccttctctga aaagcagagt cacaatttct gtggacacat ccaagaacca gttctctctg 1620

aaattgtcta gcgttaccgc cgctgataca gcagtctact attgcgctag gtcttacgga 1680

ggcaacagac tggactattg gggccaggga acactagtga cagtgagcag cggtggtggt 1740

ggaagtggtg gaggtggatc aggaggtgga ggaagtgagc tggtgctcac ccagagccct 1800

gccacactgt ccctctctcc tggcgaaagg gccacactga gctgtagagc ttcccagggc 1860

gtgggagaaa ggctgtcttg gtatcaacag aagcctggac aggctcctag attgctgatc 1920

tattactctg cctaccgcgc tactggcgtg cctgctcggt tcagtggatc tggcagcgga 1980

acagacttca ccctgacaat cagctccctg gagcctgaag actttgccgt gtactattgc 2040

cttcagacct tccacatccc ttttacattc ggccagggaa ccaagctcga aatcaag 2097

<210> 20

<211> 699

<212> PRT

<213> Artificial Sequence

<220>

<223> TPC1 amino acids

<400> 20

Gln 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 Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr

20 25 30

Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

450 455 460

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

465 470 475 480

Thr Val Ser Gly Gly Ser Ile Ser Gly Ala Tyr Met Trp Ser Trp Ile

485 490 495

Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile Val Pro

500 505 510

His Arg Ser Ala Thr Ser Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr

515 520 525

Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser

530 535 540

Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Tyr Gly

545 550 555 560

Gly Asn Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

565 570 575

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

580 585 590

Glu Leu Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

595 600 605

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Gly Glu Arg

610 615 620

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

625 630 635 640

Tyr Tyr Ser Ala Tyr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly

645 650 655

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

660 665 670

Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Thr Phe His Ile Pro Phe

675 680 685

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

690 695

<210> 21

<211> 2097

<212> DNA

<213> Artificial Sequence

<220>

<223> TPCmeutC 2 nucleotide

<400> 21

caggtgcagc ttgtggagag cggaggaggc ctggtgcagc ctggaggctc tctgagactc 60

tcctgtaccg tgtctggcat cgacctgagc tcctatgaca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagta catcggctac attagctacg tgtctcggac atactatgcc 180

gacagcgtga agggcagatt caccatctcc aaggacacct ccaagaacac ggtgtatctg 240

cagatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgccag agacaggccc 300

gatggcgctg ccaccaacct gtggggacag ggtacactag tgacagtgtc ctctgctagc 360

accaagggcc cctccgtgtt ccctctggcc ccttcttcca agtccaccag cggaggcacc 420

gccgctctgg gctgcctcgt gaaagactac ttccccgagc ccgtgaccgt gtcttggaac 480

tctggcgctc tgacctctgg cgtgcacacc ttccctgctg tgctgcagtc ctccggcctg 540

tactctctgt cctccgtcgt gactgtgccc tccagctctc tgggcaccca gacctacatc 600

tgtaacgtga accacaagcc ctccaacacc aaggtggaca agaaggtgga acctaagtct 660

tgtgacaaaa cacacacctg ccccccttgt cctgcccctg aactcctggg cggaccctct 720

gtgtttctgt tccccccaaa gcctaaggac accctgatga tctcccggac ccccgaagtg 780

acctgcgtgg tggtggatgt gtcccacgaa gatcccgagg tgaagttcaa ttggtacgtg 840

gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc 900

taccgggtgg tgtctgtgct gaccgtgctg caccaggact ggctgaacgg caaagagtac 960

aagtgcaagg tgtccaacaa ggctctgccc gcccctatcg aaaagaccat cagcaaggcc 1020

aagggccagc cccgggaacc ccaggtgtac acactgcctc caagcagaga cgagctgacc 1080

aagaatcagg tgtccctgac ctgtctcgtg aagggcttct acccctccga tatcgccgtg 1140

gaatgggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1200

tccgacggct ccttctttct gtattctaag ctgaccgtgg acaagtcccg gtggcagcag 1260

ggcaacgtgt tctcctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag 1320

tccctgtccc tgtctcccgg aaagggtggt ggaggatcag gtggtggagg aagtcaggtg 1380

cagctccagg agagcggccc tggactggtg aagccctctg agaccctcag cctgacttgc 1440

acagtgagcg gcggatctat ctctggagct tacatgtgga gctggatcag gcagcctccc 1500

ggcaagtgcc tggaatggat tgggagtatc gtgcctcaca ggagcgccac ctcctacaac 1560

ccttctctga aaagcagagt cacaatttct gtggacacat ccaagaacca gttctctctg 1620

aaattgtcta gcgttaccgc cgctgataca gcagtctact attgcgctag gtcttacgga 1680

ggcaacagac tggactattg gggccaggga acactagtga cagtgagcag cggtggtggt 1740

ggaagtggtg gaggtggatc aggaggtgga ggaagtgagc tggtgctcac ccagagccct 1800

gccacactgt ccctctctcc tggcgaaagg gccacactga gctgtagagc ttcccagggc 1860

gtgggagaaa ggctgtcttg gtatcaacag aagcctggac aggctcctag attgctgatc 1920

tattactctg cctaccgcgc tactggcgtg cctgctcggt tcagtggatc tggcagcgga 1980

acagacttca ccctgacaat cagctccctg gagcctgaag actttgccgt gtactattgc 2040

cttcagacct tccacatccc ttttacattc ggctgcggaa ccaagctcga aatcaag 2097

<210> 22

<211> 699

<212> PRT

<213> Artificial Sequence

<220>

<223> TPCmeutC 2 amino acid

<400> 22

Gln 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 Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr

20 25 30

Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

450 455 460

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

465 470 475 480

Thr Val Ser Gly Gly Ser Ile Ser Gly Ala Tyr Met Trp Ser Trp Ile

485 490 495

Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Ile Gly Ser Ile Val Pro

500 505 510

His Arg Ser Ala Thr Ser Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr

515 520 525

Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser

530 535 540

Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Tyr Gly

545 550 555 560

Gly Asn Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

565 570 575

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

580 585 590

Glu Leu Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

595 600 605

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Gly Glu Arg

610 615 620

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

625 630 635 640

Tyr Tyr Ser Ala Tyr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly

645 650 655

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

660 665 670

Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Thr Phe His Ile Pro Phe

675 680 685

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

690 695

<210> 23

<211> 2112

<212> DNA

<213> Artificial Sequence

<220>

<223> TPC5mutC3 nucleotide

<400> 23

caggtgcagc ttgtggagag cggaggaggc ctggtgcagc ctggaggctc tctgagactc 60

tcctgtaccg tgtctggcat cgacctgagc tcctatgaca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagta catcggctac attagctacg tgtctcggac atactatgcc 180

gacagcgtga agggcagatt caccatctcc aaggacacct ccaagaacac ggtgtatctg 240

cagatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgccag agacaggccc 300

gatggcgctg ccaccaacct gtggggacag ggtacactag tgacagtgtc ctctgctagc 360

accaagggcc cctccgtgtt ccctctggcc ccttcttcca agtccaccag cggaggcacc 420

gccgctctgg gctgcctcgt gaaagactac ttccccgagc ccgtgaccgt gtcttggaac 480

tctggcgctc tgacctctgg cgtgcacacc ttccctgctg tgctgcagtc ctccggcctg 540

tactctctgt cctccgtcgt gactgtgccc tccagctctc tgggcaccca gacctacatc 600

tgtaacgtga accacaagcc ctccaacacc aaggtggaca agaaggtgga acctaagtct 660

tgtgacaaaa cacacacctg ccccccttgt cctgcccctg aactcctggg cggaccctct 720

gtgtttctgt tccccccaaa gcctaaggac accctgatga tctcccggac ccccgaagtg 780

acctgcgtgg tggtggatgt gtcccacgaa gatcccgagg tgaagttcaa ttggtacgtg 840

gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc 900

taccgggtgg tgtctgtgct gaccgtgctg caccaggact ggctgaacgg caaagagtac 960

aagtgcaagg tgtccaacaa ggctctgccc gcccctatcg aaaagaccat cagcaaggcc 1020

aagggccagc cccgggaacc ccaggtgtac acactgcctc caagcagaga cgagctgacc 1080

aagaatcagg tgtccctgac ctgtctcgtg aagggcttct acccctccga tatcgccgtg 1140

gaatgggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1200

tccgacggct ccttctttct gtattctaag ctgaccgtgg acaagtcccg gtggcagcag 1260

ggcaacgtgt tctcctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag 1320

tccctgtccc tgtctcccgg aaagggtggt ggaggatcag gtggtggagg aagtcaggtg 1380

cagctccagg agagcggccc tggactggtg aagccctctg agaccctcag cctgacttgc 1440

acagtgagcg gcggatctat ctctggagct tacatgtgga gctggatcag gcagcctccc 1500

ggcaagtgcc tggaatggat tgggagtatc gtgcctcaca ggagcgccac ctcctacaac 1560

ccttctctga aaagcagagt cacaatttct gtggacacat ccaagaacca gttctctctg 1620

aaattgtcta gcgttaccgc cgctgataca gcagtctact attgcgctag gtcttacgga 1680

ggcaacagac tggactattg gggccaggga acactagtga cagtgagcag cggtggtggt 1740

ggaagtggtg gaggtggatc aggaggtgga ggaagtggag gaggaggttc agagctggtg 1800

ctcacccaga gccctgccac actgtccctc tctcctggcg aaagggccac actgagctgt 1860

agagcttccc agggcgtggg agaaaggctg tcttggtatc aacagaagcc tggacaggct 1920

cctagattgc tgatctatta ctctgcctac cgcgctactg gcgtgcctgc tcggttcagt 1980

ggatctggca gcggaacaga cttcaccctg acaatcagct ccctggagcc tgaagacttt 2040

gccgtgtact attgccttca gaccttccac atccctttta cattcggctg cggaaccaag 2100

ctcgaaatca ag 2112

<210> 24

<211> 704

<212> PRT

<213> Artificial Sequence

<220>

<223> TPC5mutC3 amino acids

<400> 24

Gln 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 Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr

20 25 30

Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

450 455 460

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

465 470 475 480

Thr Val Ser Gly Gly Ser Ile Ser Gly Ala Tyr Met Trp Ser Trp Ile

485 490 495

Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Ile Gly Ser Ile Val Pro

500 505 510

His Arg Ser Ala Thr Ser Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr

515 520 525

Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser

530 535 540

Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Tyr Gly

545 550 555 560

Gly Asn Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

565 570 575

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

580 585 590

Gly Gly Gly Gly Ser Glu Leu Val Leu Thr Gln Ser Pro Ala Thr Leu

595 600 605

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

610 615 620

Gly Val Gly Glu Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala

625 630 635 640

Pro Arg Leu Leu Ile Tyr Tyr Ser Ala Tyr Arg Ala Thr Gly Val Pro

645 650 655

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

660 665 670

Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Thr

675 680 685

Phe His Ile Pro Phe Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

690 695 700

<210> 25

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> heavy chain Signal peptide amino acids

<400> 25

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser

<210> 26

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> light chain Signal peptide amino acids

<400> 26

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys

20

<210> 27

<211> 729

<212> DNA

<213> Artificial Sequence

<220>

<223> antibody scFv nucleotide binding to PD-L1

<400> 27

caggtgcagc ttgtggagag cggaggaggc ctggtgcagc ctggaggctc tctgagactc 60

tcctgtaccg tgtctggcat cgacctgagc tcctatgaca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagta catcggctac attagctacg tgtctcggac atactatgcc 180

gacagcgtga agggcagatt caccatctcc aaggacacct ccaagaacac ggtgtatctg 240

cagatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgccag agacaggccc 300

gatggcgctg ccaccaacct gtggggacag ggtacactag tgacagtgtc ctctggtggt 360

ggtggaagtg gtggaggtgg atcaggaggt ggaggaagtg acatccagat gacacagagc 420

ccttccaccc tgtccgccag cgtgggagac agagtgacca tcacttgcca gtccagccag 480

aacgtgtact ctaacaatag actgtcatgg tatcagcaga agccagggaa ggctcctaag 540

ctcctgatct attggaccag cttcctcgcc tccggagtgc catcaaggtt cagcggcagt 600

ggatctggga cagaatttac tctgaccatc agctccctgc agcccgacga ttttgccact 660

tattactgcg ctggcggata cagcggcaac ctgtacacct tcggtcaggg taccaagttg 720

gaaattaag 729

<210> 28

<211> 243

<212> PRT

<213> Artificial Sequence

<220>

<223> antibody scFv amino acids binding to PD-L1

<400> 28

Gln 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 Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr

20 25 30

Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu

130 135 140

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln

145 150 155 160

Asn Val Tyr Ser Asn Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala

210 215 220

Gly Gly Tyr Ser Gly Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu

225 230 235 240

Glu Ile Lys

<210> 29

<211> 723

<212> DNA

<213> Artificial Sequence

<220>

<223> antibody scFv nucleotide binding to TIGIT

<400> 29

caggtgcagc tccaggagag cggccctgga ctggtgaagc cctctgagac cctcagcctg 60

acttgcacag tgagcggcgg atctatctct ggagcttaca tgtggagctg gatcaggcag 120

cctcccggca agggactgga atggattggg agtatcgtgc ctcacaggag cgccacctcc 180

tacaaccctt ctctgaaaag cagagtcaca atttctgtgg acacatccaa gaaccagttc 240

tctctgaaat tgtctagcgt taccgccgct gatacagcag tctactattg cgctaggtct 300

tacggaggca acagactgga ctattggggc cagggaacac tagtgacagt gagcagcggt 360

ggtggtggaa gtggtggagg tggatcagga ggtggaggaa gtgagctggt gctcacccag 420

agccctgcca cactgtccct ctctcctggc gaaagggcca cactgagctg tagagcttcc 480

cagggcgtgg gagaaaggct gtcttggtat caacagaagc ctggacaggc tcctagattg 540

ctgatctatt actctgccta ccgcgctact ggcgtgcctg ctcggttcag tggatctggc 600

agcggaacag acttcaccct gacaatcagc tccctggagc ctgaagactt tgccgtgtac 660

tattgccttc agaccttcca catccctttt acattcggcc agggaaccaa gctcgaaatc 720

aag 723

<210> 30

<211> 241

<212> PRT

<213> Artificial Sequence

<220>

<223> antibody scFv amino acids binding to TIGIT

<400> 30

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Gly Ala

20 25 30

Tyr Met Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Val Pro His Arg Ser Ala Thr Ser Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Tyr Gly Gly Asn Arg Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Glu Leu Val Leu Thr Gln Ser Pro Ala Thr

130 135 140

Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

145 150 155 160

Gln Gly Val Gly Glu Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ala Pro Arg Leu Leu Ile Tyr Tyr Ser Ala Tyr Arg Ala Thr Gly Val

180 185 190

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln

210 215 220

Thr Phe His Ile Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys

<210> 31

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> human IgG1 constant region amino acids

<400> 31

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 32

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker1 amino acid

<400> 32

Ala Ser Thr Lys Gly Pro

1 5

<210> 33

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker2 amino acid

<400> 33

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

1 5 10

<210> 34

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker3 amino acid

<400> 34

Thr Val Ala Ala Pro

1 5

<210> 35

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker4 amino acid

<400> 35

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

1 5 10

<210> 36

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker5 amino acid

<400> 36

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 37

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker6 amino acid

<400> 37

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 38

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker7 amino acid

<400> 38

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

Claims (12)

1. A bispecific antibody for resisting PD-L1 and TIGIT is characterized in that the bispecific antibody is an antibody TPAmutC1,

TPAmutC2, TPAmutC3, TPAmutC4, TPA5mutC1, TPCmutC2 or TPC5mutC3, comprising a light chain and a heavy chain, the heavy chain of which has the amino acid sequence shown as SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 22, SEQ ID No. 24 in sequence; the light chain is the amino acid sequence shown in SEQ ID No. 4.

2. An isolated nucleic acid encoding the bispecific antibody of claim 1.

3. An expression vector comprising the isolated nucleic acid of claim 2.

4. The expression vector of claim 3, wherein the expression vector is capable of expression in a cell line.

5. A host cell comprising the nucleic acid of claim 2.

6. A host cell comprising the expression vector of claim 3 or 4.

7. The host cell of claim 6, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

8. A method of producing a bispecific antibody, comprising culturing the host cell of claim 6 or 7, from which the bispecific antibody is expressed.

9. An immunoconjugate comprising the bispecific antibody of claim 1 and a cytotoxic agent.

10. The immunoconjugate of claim 9, wherein the immunoconjugate is a cytotoxic drug comprising a chemotherapeutic agent, a growth inhibitory agent, a toxin, or a radioisotope.

11. A pharmaceutical composition comprising the bispecific antibody of claim 1, or comprising the vector of claim 3 or 4, or comprising the immunoconjugate of claim 9 or 10, and a pharmaceutically acceptable carrier and a second therapeutic agent.

12. A kit comprising the bispecific antibody of claim 1 or the pharmaceutical composition of claim 11.