An HPV16 / 18 / 52 therapeutic vaccine, preparation method and application

Abstract

The present invention belongs to the field of bioengineering technology, in particular to an HPV16 / 18 / 52 therapeutic vaccine, a preparation method and an application. The present invention discloses a fusion protein, which is a protein of the following a) or b): a) a fusion protein with an amino acid sequence of SEQ ID No.2; b) a fusion protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID No.2 and having the activity of binding to HPV16 / 18 / 52 E6-E7. The HPV therapeutic vaccine KDTV001 designed in this application is an HPV therapeutic vaccine targeting the HPV16 / 18 / 52 E6-E7 protein with non-replicating Ad5 as the vector. The mechanism by which KDTV001 exerts an immune effect in the body after intramuscular injection is that the antigen encoded by the adenovirus vaccine is presented to T cells via antigen-presenting cells, specifically attacking the free virus of HPV16 / 18 / 52 and the cervical epithelial cells integrating the HPV16 / 18 / 52 E6 and E7 genes, ultimately clearing the virus and reversing the progression of CIN. The main application population is patients with persistent HPV infection, cervical, anal and vulvar intraepithelial neoplasia.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to an HPV16 / 18 / 52 therapeutic vaccine, a preparation method and an application thereof. Background Art

[0002] Mucosa and skin are the most common areas infected by HPV. Especially for women of childbearing age who are sexually active, they may be infected with HPV through sexual contact with an infected partner. HPV infects basal epithelial cells through damaged epithelial cells, causing cervical dysplasia and CIN. Persistent infection with high-risk HPV leads to the occurrence of cervical cancer. Most natural HPV infections are limited to the inner layer of the epithelial mucosa and do not develop into cancer. Approximately 90% of HPV-infected patients clear the virus within a few months after virus infection under the mediation of innate immune response and specific humoral immunity. 10% of patients have persistent infection, and about 1% of patients have an increased risk of developing cancer. Persistent HPV infection is an important cause ultimately leading to the occurrence of cervical cancer. Therefore, timely clearing of HPV virus infection and reversing cervical intraepithelial neoplasia are crucial for ultimately preventing the development into cervical cancer. However, there is no specific drug for HPV so far. Some traditional clinical therapies for HPV, such as laser, cryotherapy, surgery, etc., have disadvantages such as incomplete treatment, long treatment courses, and easy scarring. Among them, the most difficult to overcome is the problem of repeated recurrence of HPV after treatment. Currently, all HPV vaccines are prophylactic vaccines and have no therapeutic effect on existing infections or lesions. And existing vaccines cannot prevent persistent infections of all high-risk HPVs and the resulting cervical lesions. Even if vaccinated with an HPV vaccine, regular cervical cancer screening should be carried out according to the suggestions of relevant departments. Therefore, the development of therapeutic vaccines has broad application prospects in the treatment of HPV.

[0003] Compared with prophylactic HPV vaccines, the development of therapeutic HPV vaccines has been very slow so far. Although some research reports promising clinical responses, there is no clear relationship between inducing immune responses and clinical responses. The possible reason is that at the present stage, we lack an understanding of the immune response mechanism for controlling and clearing HPV-infected cells in the host, and the research on the tumor microenvironment and immune escape mechanism is also unclear. However, with the in-depth study of the immune mechanism against HPV infection, we are confident in designing better combinations of vaccines and adjuvants to guide the development of effective vaccines in the future and fill the gaps in the research and market of therapeutic HPV vaccines in China.

[0004] Studies have confirmed that there are significant differences in the genotype distribution and infection rates of high-risk HPV among different countries and regions, different geographical environments, different races and populations. High-risk HPV16 and HPV18 are the two genotypes with the strongest carcinogenicity identified in research. HPV16 / 18 / 58 / 33 and 52 are the five main subtypes in cervical cancer in China. The infection rate of HPV52 is particularly high in North America, Asia and Africa. Domestic research reports that the overall infection rate of HPV52 in women is as high as 2.4%, and in Central China it is even as high as 3.1%, making it the genotype with the highest infection rate among all high-risk HPVs, and its infection rate in women with cervical cancer is as high as 3.6%. Research shows that as cervical lesions worsen, the co-infection rate of high-risk HPV increases, and the multiple co-infection of HPV is associated with poor prognosis of cervical cancer, which may be related to the fact that the treatment failure rate of cervical cancer patients with multiple co-infection of HPV is higher than that of single-infected patients. Therefore, the treatment and detection of patients with multiple co-infection should be strengthened. Currently, the antigens of HPV therapeutic vaccines in clinical trials are basically all concentrated on single HPV16E6-E7 or HPV18 E6-E7, as well as the two genotypes of HPV16 and HPV18, and there is no vaccine targeting other genotypes except HPV16 and HPV18, especially HPV52 / 58 / 33 / 31, etc. Therefore, developing therapeutic HPV vaccines targeting multiple genotypes is equally important for improving the cure rate of high-grade cervical intraepithelial neoplasia.

[0005] Recently, non-replicating adenovirus vectors have once again attracted great attention as the main vectors for COVID-19 vaccines. The main advantages of non-replicating adenovirus vectors are high-titer growth ability, easy operation, non-integration into the human genome, strong immunogenicity, easy large-scale clinical production, etc. And currently, Ad vector vaccines against COVID-19 have been approved and used globally. Large-scale clinical data have shown the great potential and safety of Ad vectors as a vaccine platform for emerging and re-emerging infectious diseases. Based on the current status of HPV infection and treatment, as well as the research background of our laboratory, the designed HPV therapeutic vaccine KDTV001 is a non-replicating Ad5-based vector vaccine targeting the E6-E7 proteins of HPV16 / 18 / 52. The mechanism by which KDTV001 exerts an immune effect in the body after intramuscular injection is that the antigens encoded by the adenovirus vaccine are presented to T cells via antigen-presenting cells. Activated CD4+ cells differentiate into specific Th cells, activated CD8+ cells differentiate into specific CTL cells, and under the action of secreted cytokines such as IFN-γ and IL-4, specifically attack the free viruses of HPV16 / 18 / 52 and the cervical epithelial cells integrated with the E6 and E7 genes of HPV16 / 18 / 52, ultimately clearing the virus and reversing the progression of CIN. The main target population is patients with persistent HPV infection, cervical, anal and vulvar intraepithelial neoplasia.

[0006] SUMMARY OF THE INVENTION Based on the above object, the present invention provides a gene-optimized polynucleotide for encoding E6 and E7 fusion proteins of HPV16, HPV18 and HPV52, and the sequence of the polynucleotide is as shown in SEQ ID No.1. The polynucleotide uses an E1- and E3-deleted replication-defective human adenovirus type 5 as a vector and HEK293 cells integrating the adenovirus E1 gene as a packaging cell line, and a recombinant adenovirus vector-based novel HPV16 / 18 / 52 therapeutic vaccine is obtained through packaging. SUMMARY OF THE INVENTION

[0007] The present invention first provides a fusion protein, which is the protein of a) or b) as follows:

[0008] a) A fusion protein with an amino acid sequence of SEQ ID No.2;

[0009] b) A fusion protein with the activity of binding to HPV16 / 18 / 52 E6-E7 obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID No.2.

[0010] The present invention also provides a biomaterial related to the above fusion protein, which is any one of the following B1) to B8):

[0011] B1) A nucleic acid molecule encoding the above fusion protein;

[0012] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0013] B3) A recombinant vector containing the nucleic acid molecule described in B1),

[0014] B4) A recombinant vector containing the expression cassette described in B2);

[0015] B5) A recombinant microorganism containing the nucleic acid molecule described in B1);

[0016] B6) A recombinant microorganism containing the expression cassette described in B2);

[0017] B7) A recombinant microorganism containing the recombinant vector described in B3);

[0018] B8) A recombinant microorganism containing the recombinant vector described in B4);

[0019] Optionally, the vector includes an adenovirus vector;

[0020] Optionally, the recombinant microorganism includes HEK293A cells.

[0021] In certain embodiments, the nucleic acid molecule is the nucleic acid molecule shown in any one of 1) or 2) or 3) or 4) as follows:

[0022] 1) The coding sequence is a DNA molecule or cDNA molecule of SEQ ID No.2 in the sequence listing;

[0023] 2) The nucleic acid sequence is a DNA molecule of SEQ ID No.1 in the sequence listing;

[0024] 3) A cDNA molecule or genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) or 2) and encodes the above-mentioned protein;

[0025] 4) A cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the above-mentioned fusion protein.

[0026] The present invention also provides the use of the above-mentioned fusion protein in the preparation of a vaccine or drug for treating HPV16 / 18 / 52.

[0027] The present invention also provides the use of the above-mentioned biological material in the preparation of a vaccine or drug for treating HPV16 / 18 / 52.

[0028] The present invention also provides a primer pair for amplifying a nucleic acid molecule fragment encoding the above-mentioned fusion protein.

[0029] In some embodiments, the amplification primer pair is: the forward primer is as shown in SEQ ID NO.3, and the reverse primer is as shown in SEQ ID NO.4.

[0030] The present invention also provides a preparation method of the above-mentioned biological material, comprising the following steps: (1) cloning the HPV16 / 18 / 52 sequence; (2) performing homologous recombination on the sequence in step 1 to construct a new plasmid.

[0031] The present invention also provides a preparation method of a vaccine or drug for treating HPV16 / 18 / 52, which is obtained by cloning, propagating, and purifying using the above-mentioned biological material.

[0032] The present invention finally provides a vaccine or drug for treating HPV16 / 18 / 52, and the product comprises the above-mentioned protein or the above-mentioned biological material.

[0033] Compared with the prior art, the present invention has at least the following beneficial effects:

[0034] The HPV therapeutic vaccine KDTV001 designed in this application uses non-replicating Ad5 as a vector and is a HPV therapeutic vaccine targeting HPV16 / 18 / 52 E6-E7 proteins. The mechanism by which KDTV001 exerts an immune effect in the body after intramuscular injection is that the antigen encoded by the adenovirus vaccine is presented to T cells via antigen-presenting cells. Activated CD4+ cells differentiate into specific Th cells, activated CD8+ cells differentiate into specific CTL cells, and under the action of cytokines such as secreted IFN-γ and IL-4, specifically attack the free virus of HPV16 / 18 / 52 and the cervical epithelial cells integrated with the HPV16 / 18 / 52 E6 and E7 genes, ultimately clearing the virus and reversing the progression of CIN. The main population for application is those with persistent HPV infection, cervical, anal, and vulvar intraepithelial neoplasia. Description of the Drawings

[0035] Figure 1 Plasmid information;

[0036] Figure 2 WB detection of the expression of antigens and the expression of P53 and RB proteins after BESA-2B was transfected with KDTV001;

[0037] Figure 3 Elispot detection of the production of specific IFN-γ after KDTV001 was inoculated into C57BL / 6J mice;

[0038] Figure 4 Elispot detection of the production of specific IFN-γ after KDTV001 was inoculated into CD1 (ICR) mice;

[0039] Figure 5 Elispot detection of the production of specific IFN-γ after KDTV001 was inoculated into transgenic mice with human MHC class I molecule genotypes B-HLA-A 2.1, B-HLA-A24.2, and B-HLA-A 11.1;

[0040] Figure 6 A. Growth curve of TC-1 subcutaneous tumors after inoculation with KDTV001 and MATV0. B. In the above KDTV001 group without tumor formation, TC-1 cells were inoculated again, and the size of the tumors was observed and measured;

[0041] Figure 7 Growth inhibition curve of different doses of KDTV001 on TC-1 subcutaneous;

[0042] Figure 8 A. Specific killing effect of KDTV001 on HPV16 E7 in C57BL / 6J mice. B. Specific killing effect of KDTV001 on HPV18 E6 in C57BL / 6J mice. Detailed implementation manners

[0043] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0044] Nucleotide sequence SEQ ID No.1 of the recombinant protein:

[0045]

[0046] The amino acid sequence of the recombinant protein is as shown in SEQ ID No. 2:

[0047] MHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVGPICSQKPMHGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVGPWCAMRGDKATIKDYILDLQPETTDLHGYGQLGDSSDEEDTDGVDRPDGQAEQATSNYYIVTYCHSCDSTLRLCIHSTATDLRTLQQMLLGTLQVVGPGCARLMHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVGDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINGQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRMARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAAGHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRGQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRMFEDPATRPRTLHELCEVLEESVHEIRLQCVQCKKELQRREVYKFLFTDLRIVYRDNNPYGVGIMCLRFLSKISEYRHYQYSLYGKTLEERVKKPLSEITIRCIIGQTPLCPEEKERHVNANKRFHNIMGRWTGRCSECWRPR;

[0048] Example 1 Adenovirus construction invention technology:

[0049] The present invention designs a non-replicating type 5 adenovirus vector KDTV001 constructed by the combined application of gene synthesis technology, in vitro ligation technology and BJ5183 homologous recombination technology, specifically as follows: Based on the two plasmids Padeasy-1 and Pshuttle-CMV in the AdEasy system,

[0050] (1.1) The Pshuttle-BstBI was constructed using site-directed mutagenesis technology. The construction system was as follows: Fastmutagenesis system (Transgene, catalog number: FM111), and the primers are shown in Table 1:

[0051] Table 1 Primer sequences

[0052]

[0053] (1.2) The reaction system is shown in Table 2:

[0054] Table 2 Reaction system

[0055]

[0056] (1.3) The PCR running program was set as follows:

[0057]

[0058] (1.4) The correct clones were identified by sequencing to obtain the Pshuttle-CMV-BstBI plasmid.

[0059] (2) The Pshuttle-CMV-BstBI and Padeasy-1 plasmids were co-transfected into BJ5183 using the BJ5183 homologous recombination technology to achieve homologous recombination and obtain Pad-BstBI;

[0060] (2.1) The Pshuttle-BstBI was digested with PmeI to obtain a linearized plasmid;

[0061] (2.2) The linearized Pshuttle-BstBI was transfected into Bj5183-AD-1 competent cells, and homologous recombination was performed to obtain pad-CMV-BstBI;

[0062] (2.3) The correct clones were identified by sequencing, and the plasmids were extracted;

[0063] (2.4) It was transferred into the high-copy infection state T1, and identified again to obtain the pad-CMV-BstBI strain and plasmid. (3) In vitro gene synthesis of HPV16+18+52E6 E7 (gene sequence after codon optimization);

[0064] (4) Construct the KDTV001 plasmid:

[0065] (4.1) The pad-CMV-BstBI plasmid was digested with BstBI;

[0066] (4.2) Using HPV16+18+52 E6 E7 as a template, obtain an in vitro ligation fragment by PCR. The primers are as follows: The italic part is the PCR primer for the HPVE6E7 segment, and the underlined part is the homologous recombination overlapping segment; the primers are shown in Table 3:

[0067] Table 3 Primer Sequences

[0068]

[0069]

[0070] (4.3) Ligate the products of (4.1) and (4.2) in vitro. The reaction system is shown in Table 4:

[0071] Table 4 Reaction System

[0072]

[0073] (4.4) Transfer 5 μl of the above product into T1 for clone screening and identification;

[0074] (4.5) Obtain the KDTV001 plasmid. After digesting the KDTV001 plasmid with PacI and purifying it, transfect HEK293A cells to finally obtain the KDTV001 non-replicating adenovirus vector. See Figure 1 .

[0075] Example 2: Identify the immune effect of KDTV001.

[0076] (1) Expression of the KDTV001 antigen.

[0077] Infect BESA-2B cells in vitro at an MOI of 50. Detect the expression of the fusion protein of KDTV001 by WB. The loading order is PBS, MATV0, and KDTV001 as Figure 2 . The results show that when detected with antibodies against HPV16 E6, HPV16E7, HPV18 E, HPV18 E7, and HPV52 E7, the fusion proteins are basically at the same position, with a size of around 100 KD. Due to the lack of commercially available antibodies against HPV52 E6 and HPV52E7, the anti-HPV52 E7 antibody was synthesized by PuJian Biotech Company.

[0078] (2) Immunogenicity of KDTV001 in mice of different species.

[0079] (2.1) Immune response of KDTV001 in C57BL / 6J mice.

[0080] Intramuscularly inject KDTV001 virus with a titer of 10 8PFU / mouse. One month later, the same dose of viral booster was inoculated. The control group was the empty virus MATV0. The mice were sacrificed 1 week after the booster injection. Spleen lymphocytes were isolated, stimulated with a polypeptide pool, and the production of specific IFN-γ against the antigen by lymphocytes was detected by Elispot assay. The results are as Figure 3 shown. In the experimental group KDTV001, specific immune responses against the antigen could be generated in mice. Among them, the responses against HPV16 E6, HPV16 E7, HPV18 E6, and HPV52 E7 reached the positive standard. Most significantly, the responses against HPV16 E7 and HPV18 E6 were similar to those of the reported HPV therapeutic vaccines in C57BL / 6J mice.

[0081] (2.2) Immunogenicity of KDTV001 in CD1 (ICR) mice

[0082] Healthy 8-week-old female CD1 mice were intramuscularly injected with KDTV001 virus at a titer of 10 8 PFU / mouse. One month later, the same dose of viral booster was inoculated. The control group was the empty virus MATV0. The mice were sacrificed 1 week after the booster injection. Spleen lymphocytes were isolated, stimulated with a polypeptide pool, and the production of specific IFN-γ against the antigen by lymphocytes was detected by Elispot assay. The results are as Figure 4 shown. In the experimental group KDTV001, specific immune responses against the antigen could be generated in CD1 mice. Among them, the responses against HPV16 E6, HPV16 E7, HPV18 E7, and HPV52 E6 reached the positive standard.

[0083] (2.3) Immune responses in humanized transgenic mice

[0084] Transgenic mice with the three most common MHC class I molecular genotypes in the population, B-HLA-A2.1, B-HLA-A24.2, and B-HLA-A 11.1, were selected to verify the immunogenicity of the 6 antigens of KDTV001. After the transgenic mice were inoculated with the first dose of KDTV001 and sacrificed 1 week after the booster injection one month later, spleen lymphocytes of the mice were isolated and Elispot assay was performed to detect the specific immune responses in the mice. The results are as follows Figure 5 shown. In B-HLA-A2.1 transgenic mice, immune responses against HPV18 E6 and HPV52 E6 were generated; in B-HLA-A24.2 transgenic mice, immune responses against HPV16 E6, HPV18 E6, and HPV52 E6 were generated; in B-HLA-A11.1 transgenic mice, immune responses against HPV16 E7, HPV18 E6, HPV18 E7, and HPV52 E7 were generated.

[0085] In summary, in CD1 mice vaccinated with the vaccine, specific immune responses against HPV16 E6, HPV16 E7, HPV18 E7, and HPV52 E6 can be detected. In rats vaccinated with the vaccine, specific immune responses against HPV16 E6 and HPV18 E6 can be generated. In C57BL / 6J mice, specific immune responses against HPV16 E7 and HPV18 E6 can be detected, complementarily verifying the immunity of MAT44. In addition, in three types of MHC class I molecule humanized transgenic mice, immune responses against six antigens can be generated.

[0086] Example 3: Antitumor effect of MATV44 on TC-1 in C57BL / 6J mice.

[0087] TC-1 cells, a kind of mouse lung epithelial cells transfected with transgenic HPV16 E6, HPV16 E7, and ras genes, were used to establish a subcutaneous tumor model in the buttocks. Randomly divided into MATV44 group and MATV0 group, and vaccinated with the vaccine KDTV001 at a dose of 3×10 8 pfu / ml around the 10th day after inoculation of subcutaneous tumors, and the size of subcutaneous tumors was measured every other day and recorded. As Figure 6 shown in A, the results showed that KDTV001 could effectively inhibit the growth of TC-1 tumors at a dose of 3×10 8 pfu. One week after vaccination, the tumors in the experimental group were significantly reduced. By the end, the volume of all control group mice exceeded 2000 Mm 3 . The antitumor rate of MATV44 was 100%. A TC-1 memory model was established. TC-1 cells were inoculated again in the non-tumor-forming KDTV001 group mentioned above, and the size of the tumors was observed and measured. As Figure 6 shown in B: The memory effect of the KDTV001 vaccine could protect 100% of the mice from tumor recurrence.

[0088] (1) Correlation between the antitumor effect of the KDTV001 vaccine and the dose

[0089] A TC-1 subcutaneous tumor model in the buttocks was established, and groups were vaccinated with 10 5 , 10 6 , 5×10 6 , and 10 7 pfu / mouse of KDTV001, and the size of TC-1 tumors was measured and recorded. The tumor growth curves are as follows: The antitumor rates of 10 7 pfu, 5×10 6 pfu, and 10 6 pfu were 80%, 62%, and 31.6% respectively. See Figure 7 .

[0090] (2) In vivo killing experiment was used to detect the specific killing effect of KDTV001 on HPV16 E7 and HPV18 E6 in mice.

[0091] C57BL / 6J healthy mice were inoculated with 10 5 、10 6 、5X10 6 and 10 7 pfu / mouse of MATV44. Two weeks later, the in vivo killing experiment was performed, and the results are as follows. The killing rate of the in vivo killing experiment against the HPV16 E7 peptide was about 60% at 10 6 pfu / mouse, and the killing rate against the HPV18 E6 peptide was as high as about 90% at 10 5 pfu / mouse. See Figure 8 .

[0092] The above are the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A fusion protein, wherein the fusion protein is a fusion protein having an amino acid sequence of SEQ ID No.

2.

2. The biological material related to the fusion protein according to claim 1 is any one of the following B1) to B8): B1) a nucleic acid molecule encoding the fusion protein of claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), B4) a recombinant vector containing the expression cassette described in B2); B5) a recombinant microorganism containing the nucleic acid molecule described in B1); B6) a recombinant microorganism containing the expression cassette described in B2); B7) a recombinant microorganism containing the recombinant vector described in B3); B8) A recombinant microorganism containing the recombinant vector described in B4).

3. The biomaterial according to claim 2, wherein The vector includes an adenovirus vector.

4. The biomaterial according to claim 2, wherein The recombinant microorganism includes HEK293A cells.

5. The biomaterial according to claim 2, characterized in that: The nucleic acid molecule is the nucleic acid molecule shown in 1) or 2) below: 1) The coding sequence is a DNA molecule or cDNA molecule of SEQ ID No. 2 in the sequence list; 2) The nucleic acid sequence is the DNA molecule of SEQ ID No. 1 in the sequence list.

6. Use of the fusion protein according to claim 1 in the preparation of a vaccine or medicine for treating HPV16 / 18 / 52.

7. Use of the biomaterial according to claim 2 in the preparation of vaccines or drugs for treating HPV16 / 18 / 52.

8. A method for preparing a vaccine or drug for treating HPV16 / 18 / 52, characterized in that: The biomaterial according to claim 2 is used for cloning, propagation and purification.

9. A vaccine or medicine for treating HPV16 / 18 / 52, the product comprising the protein of claim 1 or the biological material of claim 2.

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