KR20240038901A - Novel hyaluronidase PH20 mutant and uses thereof - Google Patents

Abstract

본 발명은 신규한 히알루로니다제 PH-20 변이체 및 그 용도에 관한 것으로, 보다 상세하게는 서열번호 1, 서열번호 2, 및 서열번호 3으로 이루어진 군에서 선택된 어느 하나의 히알루로니다제 PH-20 변이체, 상기 변이체를 암호화하는 폴리뉴클레오티드, 상기 폴리펩티드를 포함하는 발현 벡터, 상기 발현 벡터를 포함하는 숙주 세포주, 상기 숙주 세포주를 배양하는 단계를 포함하는 히알루로니다제 PH-20 변이체의 제조 방법, 및 상기 히알루로니다제 PH-20 변이체를 포함하는 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다. 본 발명에 따른 신규한 히알루로니다제 PH-20 변이체는 야생형의 인간 히알루로니다제 PH-20와 비교하여 안정성이 높고 활성이 뛰어나므로, 이와 함께 사용되는 약물의 치료 효과를 극대화할 수 있다. The present invention relates to a novel hyaluronidase PH-20 variant and its use, and more specifically, to any one hyaluronidase PH- selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. 20 variant, a polynucleotide encoding the variant, an expression vector comprising the polypeptide, a host cell line comprising the expression vector, a method for producing a hyaluronidase PH-20 variant comprising culturing the host cell line, And it relates to a pharmaceutical composition for preventing or treating diseases containing the hyaluronidase PH-20 variant. The novel hyaluronidase PH-20 variant according to the present invention has high stability and excellent activity compared to wild-type human hyaluronidase PH-20, and thus can maximize the therapeutic effect of drugs used together with it.

Description

신규한 히알루로니다제 PH-20 변이체 및 그 용도{Novel hyaluronidase PH20 mutant and uses thereof}Novel hyaluronidase PH20 mutant and uses thereof}

본 발명은 신규한 히알루로니다제 PH-20 변이체 및 그 용도에 관한 것으로, 보다 상세하게는 서열번호 2, 서열번호 3, 및 서열번호 4으로 이루어진 군에서 선택된 어느 하나의 히알루로니다제 PH-20 변이체, 상기 변이체를 암호화하는 폴리뉴클레오티드, 상기 폴리펩티드를 포함하는 발현 벡터, 상기 발현 벡터를 포함하는 숙주 세포주, 상기 숙주 세포주를 배양하는 단계를 포함하는 히알루로니다제 PH-20 변이체의 제조 방법, 및 상기 히알루로니다제 PH-20 변이체를 포함하는 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a novel hyaluronidase PH-20 variant and its use, and more specifically, to any one hyaluronidase PH- selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. 20 variant, a polynucleotide encoding the variant, an expression vector comprising the polypeptide, a host cell line comprising the expression vector, a method for producing a hyaluronidase PH-20 variant comprising culturing the host cell line, And it relates to a pharmaceutical composition for preventing or treating diseases containing the hyaluronidase PH-20 variant.

히알루론산은 인간의 체내에 약 15g 정도 존재하며, 인간의 몸에서 관절 연골, 피부 조직, 눈의 유리체 등을 구성하는 주요 성분으로, 특히 세포외 기질(extracell matrix)에 풍부한 글리코사미노글리칸(glycosaminoglycan)이다. D-글루크론산(glucuronic acid)과 N-아세틸글루코사민(N-acetylglucosamine)이 β-1,4 및 β-1,3 글리코시드 결합(glycosidic bond)에 의해 교대로 연결된 이당류의 중합체로, 생체 내에서 5,000 내지 20,000,000 Da의 크기를 갖는다. 세포막에 있는 히알루론산 합성효소(hyaluronan synthase)에 의해 합성되며, 프로테오글리칸(proteoglycan)과 결합하지 않고 단독으로 존재하는, 황산기가 없는 유일한 글리코사미노글리칸이다. Hyaluronic acid exists in the human body in approximately 15g, and is a major component of joint cartilage, skin tissue, and vitreous body of the eye, and is particularly rich in glycosaminoglycans (extracell matrix). It is glycosaminoglycan). It is a polymer of disaccharides in which D-glucuronic acid and N-acetylglucosamine are alternately linked by β-1,4 and β-1,3 glycosidic bonds. It has a size of 5,000 to 20,000,000 Da. It is synthesized by hyaluronan synthase in the cell membrane, and is the only glycosaminoglycan without a sulfate group that exists alone without combining with proteoglycan.

히알루로니다제(hyaluronidase)는 히알루론산(hyaluronic acid)을 분해하는 효소이다. 인간에게는 Hyal1, Hyal2, Hyal3, Hyal4, 및 Hyal5 (PH-20 또는 SPAM1으로도 알려짐) 5 종류의 히알루로니다제가 있으며, 여기에 더하여 pseudogene으로서 발현되지 않는 Hyal6P (Hyalp1)가 있는 것으로 알려져 있다. Hyal1, Hyal2, 및 Hyal3 유전자는 3번 염색체에 존재하며, Hyal4, Hyal5, 및 Hyal6P 유전자는 7번 염색체에 존재하고 있다. 인간에서 Hyal1 및 Hyal2는 대부분의 조직에서 발현되는 히알루로니다제이며 pH 3~4 에서 최적의 활성을 나타낸다. 이와 달리, PH-20은 정자의 세포막과 첨체막에서 발현되며, pH 3~8의 넓은 구간에서 활성을 갖는다. Hyaluronidase is an enzyme that decomposes hyaluronic acid. In humans, there are five types of hyaluronidases: Hyal1, Hyal2, Hyal3, Hyal4, and Hyal5 (also known as PH-20 or SPAM1), and in addition, Hyal6P (Hyalp1), which is not expressed as a pseudogene, is known to exist. Hyal1, Hyal2, and Hyal3 genes exist on chromosome 3, and Hyal4, Hyal5, and Hyal6P genes exist on chromosome 7. In humans, Hyal1 and Hyal2 are hyaluronidases expressed in most tissues and show optimal activity at pH 3-4. In contrast, PH-20 is expressed in the cell membrane and acrosome membrane of sperm and is active in a wide pH range of 3 to 8.

PH-20은 Lathrop 등에 의해 기니피그의 정자에서 처음 동정되었고, 다른 많은 종의 정자에도 발현됨이 알려져있다. 인간의 PH-20 유전자는 SPAM1 (sperm ahdesion molecule 1) 유전자로부터 코딩되며 정자의 세포막 표면과 첨체의 막 안쪽에 PH-20의 Ser490이 glycosylphophatidylinositol (GPI)과 결합한 형태로 존재한다. 정자의 PH-20은 히알루론산이 많은 난자의 cumulus layer를 통과하여 난자 안으로 침투할 때, 히알루론산을 가수분해하는 역할을 한다.PH-20 was first identified in guinea pig sperm by Lathrop et al., and is known to be expressed in sperm of many other species. The human PH-20 gene is encoded from the SPAM1 (sperm ahdesion molecule 1) gene, and exists in the form of Ser490 of PH-20 bound to glycosylphophatidylinositol (GPI) on the surface of the sperm cell membrane and inside the acrosome membrane. PH-20 of sperm plays a role in hydrolyzing hyaluronic acid when it passes through the cumulus layer of an egg rich in hyaluronic acid and penetrates into the egg.

또한, PH-20은 중성 pH에서도 활성을 갖는다는 그 특수성 때문에 치료 약물과 함께 투여되기도 한다. 즉, 고용량 또는 다량 투여가 필요한 약물들, 특히 항체 의약품 등은 일반적으로 정맥 주사를 통하여 투여되는 것이 일반적인데, 이 경우 주사 시간만 약 90분 이상 소요되고, 정맥 주사를 위한 추가적인 조제 작업이 수반되어 환자와 의료진 모두에게 불편함을 발생시키며, 추가적인 비용이 수반되는 문제점이 있다. 이에 반하여 피하주사는 즉각적인 투여가 가능하다는 장점이 있으나, 정맥 주사 대비 흡수율이 낮고, 흡수가 느리게 일어나므로 주사량이 3~5mL 이상일 경우 주사 부위에 팽윤과 통증을 유발할 수 있다. 이러한 이유로 단백질 치료제의 피하주사는 통상 2mL 이하의 소량으로 한정하여 사용되었다. 그러나 히알루로니다제인 PH-20을 치료 약물과 함께 피하 투여하면 히알루로니다제의 작용에 의해 피부 세포외 기질에 분포하는 히알루론산이 가수분해되어 피하부의 점성이 감소하고 물질 투과성이 증가하므로, 고용량의 의약품을 체내에 용이하게 전달할 수 있다. Additionally, PH-20 is sometimes administered together with therapeutic drugs due to its unique property of being active even at neutral pH. In other words, drugs that require high or large doses, especially antibody drugs, are generally administered through intravenous injection. In this case, the injection time alone takes about 90 minutes or more, and additional preparation work for intravenous injection is involved. There is a problem that it causes inconvenience to both patients and medical staff, and involves additional costs. In contrast, subcutaneous injection has the advantage of allowing immediate administration, but the absorption rate is lower than that of intravenous injection, and absorption occurs slowly, so if the injection amount is more than 3 to 5 mL, it may cause swelling and pain at the injection site. For this reason, subcutaneous injection of protein therapeutics was usually limited to small amounts of 2 mL or less. However, when PH-20, a hyaluronidase, is administered subcutaneously together with a therapeutic drug, hyaluronic acid distributed in the skin extracellular matrix is hydrolyzed by the action of hyaluronidase, thereby reducing the viscosity of the subcutaneous area and increasing substance permeability. Medicines can be easily delivered into the body.

따라서, PH-20을 이용하여 다양한 치료용 의약품을 피하주사의 제형으로 개발하려는 연구가 진행되고 있다. 현재 상업적으로 많이 이용되는 PH-20은 소나 양의 고환에서 추출한 것이나, 동물 유래 히알루로니다제를 인체에 고용량으로 반복 투여할 경우, 중화 항체가 생성될 수 있으며, PH-20 이외에 불순물로 포함된 동물 유래의 다른 물질이 알러지를 유발할 위험도 있다. 이러한 문제를 개선하기 위해 인간 PH-20 단백질을 이용한 연구가 진행되고 있는데, 다만, 인간 PH-20 자체의 안정성이 낮기 때문에 이를 먼저 극복할 필요가 있다.Therefore, research is underway to develop various therapeutic drugs in a subcutaneous injection formulation using PH-20. PH-20, which is currently widely used commercially, is extracted from the testicles of cows or sheep. However, when animal-derived hyaluronidase is repeatedly administered to the human body in high doses, neutralizing antibodies may be generated, and in addition to PH-20, it may contain impurities. There is also a risk that other substances of animal origin may cause allergies. To improve this problem, research using human PH-20 protein is in progress, but since the stability of human PH-20 itself is low, it is necessary to overcome this first.

이를 위하여 대한민국 공개특허 제10-2020-0017538호 등에서는 인간 야생형 PH-20 단백질에 다수의 돌연변이를 도입하여 히알루로니다제의 효소 활성과 열 안정성을 높인 PH-20 변이체를 제시하였다. 그러나, 단백질에 많은 돌연변이를 도입하게 되면 면역 항원성 문제를 야기할 가능성도 함께 높아지는 문제가 있다. 따라서 야생형 PH-20에 도입되는 돌연변이는 최소화 하면서도 단백질 안정성과 히알루로니다제 활성은 극대화한 인간 유래 PH-20 변이체 개발이 필요한 실정이다.To this end, Republic of Korea Patent Publication No. 10-2020-0017538, etc. proposed a PH-20 variant in which the enzymatic activity and thermal stability of hyaluronidase were increased by introducing a number of mutations into the human wild-type PH-20 protein. However, there is a problem that introducing many mutations into a protein also increases the possibility of causing immunogenicity problems. Therefore, there is a need to develop human-derived PH-20 variants that maximize protein stability and hyaluronidase activity while minimizing mutations introduced into wild-type PH-20.

이에 본 발명자들은 인간 유래 야생형 PH-20 단백질에 도입되는 돌연변이를 최소한으로 줄여서 면역원성 문제를 최소화하였으며, 단백질의 내부에 위치하는 아미노산들의 돌연변이와 공유결합을 통해 단백질의 안정성은 높이고 효소 활성은 극대화한 신규한 PH-20 변이체를 개발함으로써 본 발명을 완성하였다.Accordingly, the present inventors minimized the immunogenicity problem by minimizing mutations introduced into the human-derived wild-type PH-20 protein, and increased protein stability and maximized enzyme activity through mutations and covalent bonds of amino acids located inside the protein. The present invention was completed by developing a novel PH-20 variant.

따라서, 본 발명의 목적은 서열번호 2, 서열번호 3, 및 서열번호 4으로 이루어진 군에서 선택되며, 추가로 변이가 0 개 내지 3 개 도입된 히알루로니다제 PH-20 변이체를 제공하는 것이다. Therefore, the object of the present invention is to provide a hyaluronidase PH-20 variant selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and in which 0 to 3 mutations are additionally introduced.

본 발명의 다른 목적은 상기 히알루로니다제 PH-20 변이체를 암호화하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 발현 벡터, 상기 발현 벡터를 포함하는 숙주 세포주, 및 상기 숙주 세포주를 배양하는 단계를 포함하는 히알루로니다제 PH-20 변이체의 제조 방법을 제공하는 것이다.Another object of the present invention is a polynucleotide encoding the hyaluronidase PH-20 variant, an expression vector comprising the polynucleotide, a host cell line comprising the expression vector, and culturing the host cell line. To provide a method for producing hyaluronidase PH-20 variant.

본 발명의 또 다른 목적은 a) 약물; 및 b) 상기 히알루로니다제 PH-20 변이체;를 포함하는 것을 특징으로 하는 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is a) drug; and b) the hyaluronidase PH-20 variant; to provide a pharmaceutical composition for preventing or treating a disease, comprising:

상기와 같은 목적을 달성하기 위하여, 본 발명은 서열번호 2, 서열번호 3, 및 서열번호 4으로 이루어진 군에서 선택되며, 추가로 변이가 0 개 내지 3 개 도입된 히알루로니다제 PH-20 변이체를 제공한다. In order to achieve the above object, the present invention provides a hyaluronidase PH-20 variant selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and in which 0 to 3 mutations are additionally introduced. provides.

본 발명의 다른 목적을 달성하기 위하여, 본 발명은 상기 히알루로니다제 PH-20 변이체를 암호화하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 발현 벡터, 상기 발현 벡터를 포함하는 숙주 세포주, 및 상기 숙주 세포주를 배양하는 단계를 포함하는 히알루로니다제 PH-20 변이체의 제조 방법을 제공한다.In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding the hyaluronidase PH-20 variant, an expression vector containing the polynucleotide, a host cell line containing the expression vector, and the host cell line. Provides a method for producing a hyaluronidase PH-20 variant comprising the step of culturing.

본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 a) 약물; 및 b) 상기 히알루로니다제 PH-20 변이체;를 포함하는 것을 특징으로 하는 질환의 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a) a drug; and b) the hyaluronidase PH-20 variant. It provides a pharmaceutical composition for preventing or treating diseases, comprising:

이하 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.

본 발명은 서열번호 2, 서열번호 3, 및 서열번호 4으로 이루어진 군에서 선택된 어느 하나의 히알루로니다제 PH-20 변이체를 제공한다. The present invention provides any one hyaluronidase PH-20 variant selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

인간 야생형 PH-20 단백질은 하기 서열번호 1의 아미노산 서열을 갖는 단백질로서, 아직 단백질 구조가 밝혀지지 않았다. 인간 히알루로니다제 중에서 단백질 구조가 밝혀진 것은 Hyal1으로, Hyal1과 PH-20의 아미노산 서열은 35.1% 일치한다. The human wild-type PH-20 protein is a protein with the amino acid sequence shown in SEQ ID NO: 1, and the protein structure has not yet been revealed. Among human hyaluronidase, the protein structure for which the protein structure was revealed is Hyal1, and the amino acid sequences of Hyal1 and PH-20 are 35.1% identical.

서열번호 1. 인간 야생형 PH-20 아미노산 서열SEQ ID NO: 1. Human wild type PH-20 amino acid sequence

MGVLKFKHIF FRSFVKSSGV SQIVFTFLLI PCCLTLNFRA PPVIPNVPFL WAWNAPSEFC MGVLKFKHIF FRSFVKSSGV SQIVFTFLLI PCCLTLNFRA PPVIPNVPFL WAWNAPSEFC

LGKFDEPLDM SLFSFIGSPR INATGQGVTI FYVDRLGYYP YIDSITGVTV NGGIPQKISLLGKFDEPLDM SLFSFIGSPR INATGQGVTI FYVDRLGYP YIDSITGVTV NGGIPQKISL

QDHLDKAKKD ITFYMPVDNL GMAVIDWEEW RPTWARNWKP KDVYKNRSIE LVQQQNVQLS QDHLDKAKKD ITFYMPVDNL GMAVIDWEEW RPTWARNWKP KDVYKNRSIE LVQQQNVQLS

LTEATEKAKQ EFEKAGKDFL VETIKLGKLL RPNHLWGYYL FPDCYNHHYK KPGYNGSCFN LTEATEKAKQ EFEKAGKDFL VETIKLGKLL RPNHLWGYYL FPDCYNHHYK KPGYNGSCFN

VEIKRNDDLS WLWNESTALY PSIYLNTQQS PVAATLYVRN RVREAIRVSK IPDAKSPLPV VEIKRNDDLS WLWNESTALY PSIYLNTQQS PVAATLYVRN RVREAIRVSK IPDAKSPLPV

FAYTRIVFTD QVLKFLSQDE LVYTFGETVA LGASGIVIWG TLSIMRSMKS CLLLDNYMET FAYTRIVFTD QVLKFLSQDE LVYTFGETVA LGASGIVIWG TLSIMRSMKS CLLLDNYMET

ILNPYIINVT LAAKMCSQVL CQEQGVCIRK NWNSSDYLHL NPDNFAIQLE KGGKFTVRGK ILNPYIINVT LAAKMCSQVL CQEQGVCIRK NWNSSDYLHL NPDNFAIQLE KGGKFTVRGK

PTLEDLEQFS EKFYCSCYST LSCKEKADVK DTDAVDVCIA DGVCIDAFLK PPMETEEPQI PTLEDLEQFS EKFYCSCYST LSCKEKADVK DTDAVDVCIA DGVCIDAFLK PPMETEEPQI

FYNASPSTLS ATMFIVSILF LIISSVASLFYNASPSTLS ATMFIVSILF LIISSVASL

본 발명자는 상기 인간 야생형 PH-20의 아미노산 서열에서, 신호 서열(signal peptide)인 1 내지 35번째 아미노산 잔기와, C-말단의 일부 서열이 잘린 서열번호 2의 Hy1 단백질을 먼저 제작하였으며, N-말단 및 C-말단의 변형이 효소 활성에 영향을 줄 수 있다고 판단하여, Hy1 아미노산 서열에서 C-말단에 아스파라긴(asparagine)이 추가된 Hy2 (서열번호 3) 및 Hy2 아미노산 서열에서 N-말단에 2개의 아미노산 잔기, 류신(leucine)과 아스파라긴(asparagine)이 삭제된 Hy3 (서열번호 4) 단백질을 추가로 제작하였다. The present inventor first produced the Hy1 protein of SEQ ID NO: 2, in which amino acid residues 1 to 35, which are the signal peptide, and part of the C-terminal sequence were cut from the amino acid sequence of human wild-type PH-20, and N- It was determined that modification of the terminal and C-terminus may affect enzyme activity, so Hy2 (SEQ ID NO: 3) with asparagine added to the C-terminus in the Hy1 amino acid sequence and 2 to the N-terminus in the Hy2 amino acid sequence. Hy3 (SEQ ID NO: 4) protein with the amino acid residues leucine and asparagine deleted was additionally produced.

본 발명에 있어, 각 변이체의 아미노산 잔기 위치는 서열번호 2에 따른 Hy1의 아미노산 위치에 따른다.In the present invention, the amino acid residue position of each variant follows the amino acid position of Hy1 according to SEQ ID NO: 2.

상기 서열번호 2의 아미노산 서열을 바탕으로 active site 모델링과 이황화 결합(disulfide bond) 모델링 과정을 거쳐 추가적인 돌연변이를 도입하였다. Based on the amino acid sequence of SEQ ID NO: 2, additional mutations were introduced through active site modeling and disulfide bond modeling processes.

Active site 모델링을 위하여 단백질 3차 구조 모델링은 DeepMind 社의 알파폴드2 (AlphaFold 2)와 (주)굿인텔리전스의 CSA (conformational space annealing) 모델링 기법을 사용하여 3차 구조를 모델링 하였다. Energy minimization은 GROMACS package를 사용하여 local energy minimization을 하였고, CHARMM36 force field를 하였다. PH-20 모델링 구조로부터 여러 변이체를 만들기 위해 Chimera, PyMOL software를 사용하였다. 또한 각 변이체의 단백질-리간드 결합력 예측을 위해 여러 종류의 예측 프로그램을 사용하였다.For active site modeling, the protein tertiary structure was modeled using DeepMind's AlphaFold 2 and Good Intelligence's CSA (conformational space annealing) modeling technique. Energy minimization was performed using the GROMACS package for local energy minimization and CHARMM36 force field. Chimera and PyMOL software were used to create several variants from the PH-20 modeling structure. In addition, several types of prediction programs were used to predict the protein-ligand binding ability of each variant.

이황화 결합 모델링을 위하여, PDB에 등록된 단백질의 구조를 이용하여 이황화 결합이 가능한 후보군을 선정한 뒤, protein contact map visualization 프로그램을 이용하여 2개의 아미노산의 C-alpha 탄소의 거리가 6Å 이하, C-beta 탄소의 거리가 5Å인 잔기를 plot을 이용하여 분석하였다. 이후 YASARA Web Energy Minimization Server를 이용하여 이황화 결합의 생성 유무를 분석한 뒤 Chimera의 AMBER force filed FF99를 이용하여 energy minimization을 진행하였다. 이후 생성된 단백질의 구조를 야생형의 PH-20과 align 하여 RMSD 측정 값이 0.5 이하를 갖도록 하는 돌연변이를 선별하였다.For disulfide bond modeling, candidates with possible disulfide bonds were selected using the structure of the protein registered in the PDB, and then using a protein contact map visualization program, the distance between the C-alpha carbons of the two amino acids was 6Å or less, and the C-beta carbon was 6Å or less. Residues with a carbon distance of 5Å were analyzed using a plot. Afterwards, the presence or absence of disulfide bonds was analyzed using YASARA Web Energy Minimization Server, and then energy minimization was performed using Chimera's AMBER force field FF99. Afterwards, the structure of the produced protein was aligned with the wild type PH-20, and mutations that had an RMSD measurement value of 0.5 or less were selected.

이에 따라 본 발명의 일 실시 양태에서는, 상기 서열번호 2, 서열번호 3, 및 서열번호 4로 이루어진 군에서 선택된 어느 하나의 히알루로니다제 PH-20 변이체에서, ⅰ) Y57H, ⅱ) W304H, ⅲ) T306R, ⅵ) L307K, 및 ⅴ) T306A로 이루어진 군에서 선택된 어느 하나의 아미노산 잔기의 치환을 더 포함하는 것을 특징으로 하는 히알루로니다제 PH-20 변이체를 제공한다.Accordingly, in one embodiment of the present invention, in any one hyaluronidase PH-20 variant selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, i) Y57H, ii) W304H, iii ) T306R, vi) L307K, and v) T306A. Provided is a hyaluronidase PH-20 variant characterized in that it further comprises a substitution of any one amino acid residue selected from the group consisting of.

본 발명의 다른 일 실시 양태에서는, 상기 서열번호 2, 서열번호 3, 및 서열번호 4로 이루어진 군에서 선택된 어느 하나의 히알루로니다제 PH-20 변이체에서, ⅰ) Y57H, ⅱ) W304H, ⅲ) T306R, 및 ⅵ) L307K로 이루어진 군에서 선택된 어느 하나의 아미노산 잔기의 치환을 더 포함하는 것을 특징으로 하는 히알루로니다제 PH-20 변이체를 제공한다.In another embodiment of the present invention, in any one hyaluronidase PH-20 variant selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, i) Y57H, ii) W304H, iii) Provided is a hyaluronidase PH-20 variant, characterized in that it further comprises the substitution of any one amino acid residue selected from the group consisting of T306R, and vi) L307K.

본 발명의 또 다른 일 실시 양태에서는, 상기 서열번호 2, 서열번호 3, 및 서열번호 4로 이루어진 군에서 선택된 어느 하나의 히알루로니다제 PH-20 변이체에서, ⅰ) P80C 및 A160C, ⅱ) G291C 및 Y362C, ⅲ) L180C 및 A223C, ⅵ) P6C 및 Y403C, 및 ⅴ) S36C 및 D426C로 이루어진 군에서 선택된 어느 하나의 아미노산 잔기의 치환을 더 포함하는 것을 특징으로 하는 히알루로니다제 PH-20 변이체를 제공한다.In another embodiment of the present invention, in any one hyaluronidase PH-20 variant selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, i) P80C and A160C, ii) G291C and Y362C, iii) L180C and A223C, vi) P6C and Y403C, and v) S36C and D426C. to provide.

본 발명의 또 다른 일 실시 양태에서는, 상기 서열번호 2, 서열번호 3, 및 서열번호 4로 이루어진 군에서 선택된 어느 하나의 히알루로니다제 PH-20 변이체에서, ⅰ) G291C 및 Y362C, ⅱ) L180C 및 A223C, ⅲ) P6C 및 Y403C, 및 ⅵ) S36C 및 D426C로 이루어진 군에서 선택된 어느 하나의 아미노산 잔기의 치환을 더 포함하는 것을 특징으로 하는 히알루로니다제 PH-20 변이체를 제공한다.In another embodiment of the present invention, in any one hyaluronidase PH-20 variant selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, i) G291C and Y362C, ii) L180C and A223C, iii) P6C and Y403C, and vi) S36C and D426C.

본 발명의 또 다른 일 실시 양태에서는, 상기 서열번호 2, 서열번호 3, 및 서열번호 4로 이루어진 군에서 선택된 어느 하나의 히알루로니다제 PH-20 변이체, 또는 ⅰ) Y57H, ⅱ) W304H, ⅲ) T306R, ⅵ) L307K, 및 ⅴ) T306A로 이루어진 군에서 선택된 어느 하나의 아미노산 잔기의 치환을 더 포함하는 것을 특징으로 하는 히알루로니다제 PH-20 변이체에서, ⅰ) P80C 및 A160C, ⅱ) G291C 및 Y362C, ⅲ) L180C 및 A223C, ⅵ) P6C 및 Y403C, 및 ⅴ) S36C 및 D426C로 이루어진 군에서 선택된 어느 하나의 아미노산 잔기의 치환을 더 포함하는 것을 특징으로 하는 히알루로니다제 PH-20 변이체를 제공한다. In another embodiment of the present invention, any one hyaluronidase PH-20 variant selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, or i) Y57H, ii) W304H, iii) ) T306R, vi) L307K, and v) T306A in the hyaluronidase PH-20 variant, i) P80C and A160C, ii) G291C. and Y362C, iii) L180C and A223C, vi) P6C and Y403C, and v) S36C and D426C. to provide.

본 발명에 있어서, "Y57"과 같이 1 글자 아미노산 잔기 명과 숫자가 함께 기재된 표현은 서열번호 2에 따른 아미노산 서열에서의 각 위치에서 아미노산 잔기를 의미한다. 예를 들어 "Y57"은 서열번호 2의 아미노산 서열에서의 57번째 위치의 아미노산 잔기가 티로신임을 의미한다.In the present invention, an expression such as "Y57" written together with a one-letter amino acid residue name and a number refers to the amino acid residue at each position in the amino acid sequence according to SEQ ID NO: 2. For example, “Y57” means that the amino acid residue at position 57 in the amino acid sequence of SEQ ID NO: 2 is tyrosine.

또한, "Y57H"는 서열번호 2의 57번째 티로신이 히스티딘으로 치환된 것임을 의미한다. Additionally, “Y57H” means that the 57th tyrosine in SEQ ID NO: 2 is replaced with histidine.

본 발명에 따른 PH-20 변이체는 특정 아미노산 잔기 위치에서 아미노산 잔기가 보존적으로 치환된 변이체를 포함하는 의미로 해석된다. PH-20 variants according to the present invention are interpreted to include variants in which amino acid residues are conservatively substituted at specific amino acid residue positions.

상기 '보존적 치환'이란 1개 이상의 아미노산을 PH-20 변이체의 생물학적 또는 생화학적 기능의 손실을 야기하지 않는 유사한 생화학적 특성을 갖는 아미노산으로 치환하는 것을 포함하는 PH-20 변이체의 변형을 의미한다. The term 'conservative substitution' refers to a modification of a PH-20 variant that includes substituting one or more amino acids with an amino acid having similar biochemical properties that does not cause loss of biological or biochemical functions of the PH-20 variant.

'보존적 아미노산 치환'은 아미노산 잔기를 유사한 측쇄를 갖는 아미노산 잔기로 대체시키는 치환이다. 유사한 측쇄를 갖는 아미노산 잔기 부류는 해당 기술분야에 잘 알려져 있다. 이들 부류는 염기성 측쇄를 갖는 아미노산(예를 들어, 라이신, 아르기닌, 히스티딘), 산성 측쇄를 갖는 아미노산(예를 들어, 아스파르트산, 글루탐산), 대전되지 않은 극성 측쇄를 갖는 아미노산(예를 들어, 글리신, 아스파라긴, 글루타민, 세린, 트레오닌, 티로신, 시스테인), 비-극성 측쇄를 갖는 아미노산(예를 들어, 알라닌, 발린, 류신, 이소류신, 프롤린, 페닐알라닌, 메티오닌, 트립토판), 베타-분지된 측쇄를 갖는 아미노산(예를 들어, 트레오닌, 발린, 이소류신) 및 방향족 측쇄를 갖는 아미노산(예를 들어, 티로신, 페닐알라닌, 트립토판, 히스티딘)을 포함한다.'Conservative amino acid substitution' is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Classes of amino acid residues with similar side chains are well known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), and amino acids with uncharged polar side chains (e.g., glycine). , asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains Includes amino acids (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

본 발명에 있어서 PH-20 변이체는 보존적 아미노산 치환을 갖더라도 여전히 활성을 보유할 수 있음이 예상된다. 또한, 본 발명에 따른 PH-20 변이체는, 본 발명에 따른 PH-20 변이체와 실질적으로 동일한 기능 및/또는 효과를 가지며, 80% 또는 85% 이상, 바람직하게는 90% 이상, 더욱 바람직하게는 95% 이상, 가장 바람직하게는 99% 이상의 아미노산 서열 상동성을 가지는 PH-20 변이체도 포함하는 의미로 해석된다.In the present invention, it is expected that the PH-20 variant may still retain activity even if it has conservative amino acid substitutions. In addition, the PH-20 variant according to the present invention has substantially the same function and/or effect as the PH-20 variant according to the present invention, and is preferably 80% or 85% or more, preferably 90% or more, and more preferably 90% or more. It is interpreted to include PH-20 variants having amino acid sequence homology of 95% or more, most preferably 99% or more.

바람직하게는, 본 발명에 따른 히알루로니다제 PH-20 변이체는 서열번호 2 내지 서열번호 109의 아미노산 서열로 구성된 군에서 선택된 어느 하나일 수 있다. 본 발명에 따른 실시예에서 제작한 히알루로니다제 PH-20 변이체의 아미노산 서열은 하기의 표 1에 기재된 바와 같다.Preferably, the hyaluronidase PH-20 variant according to the present invention may be any one selected from the group consisting of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 109. The amino acid sequence of the hyaluronidase PH-20 variant produced in the examples according to the present invention is as shown in Table 1 below.

서열
번호order
number 명칭designation 돌연변이mutation 아미노산 서열amino acid sequence 22 Hy1Hy1 　 LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 33 Hy2Hy2 　 LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 44 Hy3Hy3 　 FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 55 Hy1-M1Hy1-M1 P80C, A160CP80C, A160C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 66 Hy1-M2Hy1-M2 G291C, Y362CG291C, Y362C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 77 Hy1-M3Hy1-M3 L180C, A223CL180C, A223C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 88 Hy1-M4Hy1-M4 P6C, Y403CP6C, Y403C LNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 99 Hy1-M5Hy1-M5 S36C, D426CS36C, D426C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFY 1010 Hy1-A1Hy1-A1 Y57HY57H LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 1111 Hy1-A1-M1Hy1-A1-M1 Y57H, P80C, A160CY57H, P80C, A160C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 1212 Hy1-A1-M2Hy1-A1-M2 Y57H, G291C, Y362CY57H, G291C, Y362C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 1313 Hy1-A1-M3Hy1-A1-M3 Y57H, L180C, A223CY57H, L180C, A223C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 1414 Hy1-A1-M4Hy1-A1-M4 Y57H, P6C, Y403CY57H, P6C, Y403C LNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 1515 Hy1-A1-M5Hy1-A1-M5 Y57H, S36C, D426CY57H, S36C, D426C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFY 1616 Hy1-A2Hy1-A2 W304HW304H LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 1717 Hy1-A2-M1Hy1-A2-M1 W304H, P80C, A160CW304H, P80C, A160C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 1818 Hy1-A2-M2Hy1-A2-M2 W304H, G291C, Y362CW304H, G291C, Y362C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKE KADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 1919 Hy1-A2-M3Hy1-A2-M3 W304H, L180C, A223CW304H, L180C, A223C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 2020 Hy1-A2-M4Hy1-A2-M4 W304H, P6C, Y403CW304H, P6C, Y403C LNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCCSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 2121 Hy1-A2-M5Hy1-A2-M5 W304H, S36C, D426CW304H, S36C, D426C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFY 2222 Hy1-A3Hy1-A3 T306RT306R LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 2323 Hy1-A3-M1Hy1-A3-M1 T306R, P80C, A160CT306R, P80C, A160C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 2424 Hy1-A3-M2Hy1-A3-M2 T306R, G291C, Y362CT306R, G291C, Y362C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 2525 Hy1-A3-M3Hy1-A3-M3 T306R, L180C, A223CT306R, L180C, A223C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 2626 Hy1-A3-M4Hy1-A3-M4 T306R, P6C, Y403CT306R, P6C, Y403C LNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 2727 Hy1-A3-M5Hy1-A3-M5 T306R, S36C, D426CT306R, S36C, D426C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFY 2828 Hy1-A4Hy1-A4 L307KL307K LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 2929 Hy1-A4-M1Hy1-A4-M1 L307K, P80C, A160CL307K, P80C, A160C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 3030 Hy1-A4-M2Hy1-A4-M2 L307K, G291C, Y362CL307K, G291C, Y362C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 3131 Hy1-A4-M3Hy1-A4-M3 L307K, L180C, A223CL307K, L180C, A223C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 3232 Hy1-A4-M4Hy1-A4-M4 L307K, P6C, Y403CL307K, P6C, Y403C LNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 3333 Hy1-A4-M5Hy1-A4-M5 L307K, S36C, D426CL307K, S36C, D426C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFY 3434 Hy1-A5Hy1-A5 T306AT306A LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 3535 Hy1-A5-M1Hy1-A5-M1 T306A, P80C, A160CT306A, P80C, A160C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 3636 Hy1-A5-M2Hy1-A5-M2 T306A, G291C, Y362CT306A, G291C, Y362C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 3737 Hy1-A5-M3Hy1-A5-M3 T306A, L180C, A223CT306A, L180C, A223C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 3838 Hy1-A5-M4Hy1-A5-M4 T306A, P6C, Y403CT306A, P6C, Y403C LNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYLNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY 3939 Hy1-A5-M5Hy1-A5-M5 T306A, S36C, D426CT306A, S36C, D426C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFY 4040 Hy2-M1Hy2-M1 P80C, A160CP80C, A160C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 4141 Hy2-M2Hy2-M2 G291C, Y362CG291C, Y362C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 4242 Hy2-M3Hy2-M3 L180C, A223CL180C, A223C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 4343 Hy2-M4Hy2-M4 P6C, Y403CP6C,Y403C LNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 4444 Hy2-M5Hy2-M5 S36C, D426CS36C, D426C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYN 4545 Hy2-A1Hy2-A1 Y57HY57H LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 4646 Hy2-A1-M1Hy2-A1-M1 Y57H, P80C, A160CY57H, P80C, A160C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 4747 Hy2-A1-M2Hy2-A1-M2 Y57H, G291C, Y362CY57H, G291C, Y362C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 4848 Hy2-A1-M3Hy2-A1-M3 Y57H, L180C, A223CY57H, L180C, A223C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 4949 Hy2-A1-M4Hy2-A1-M4 Y57H, P6C, Y403CY57H, P6C, Y403C LNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 5050 Hy2-A1-M5Hy2-A1-M5 Y57H, S36C, D426CY57H, S36C, D426C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYN 5151 Hy2-A2Hy2-A2 W304HW304H LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 5252 Hy2-A2-M1Hy2-A2-M1 W304H, P80C, A160CW304H, P80C, A160C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 5353 Hy2-A2-M2Hy2-A2-M2 W304H, G291C, Y362CW304H, G291C, Y362C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKE KADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 5454 Hy2-A2-M3Hy2-A2-M3 W304H, L180C, A223CW304H, L180C, A223C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 5555 Hy2-A2-M4Hy2-A2-M4 W304H, P6C, Y403CW304H, P6C, Y403C LNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCCSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 5656 Hy2-A2-M5Hy2-A2-M5 W304H, S36C, D426CW304H, S36C, D426C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYN 5757 Hy2-A3Hy2-A3 T306RT306R LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 5858 Hy2-A3-M1Hy2-A3-M1 T306R, P80C, A160CT306R, P80C, A160C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 5959 Hy2-A3-M2Hy2-A3-M2 T306R, G291C, Y362CT306R, G291C, Y362C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 6060 Hy2-A3-M3Hy2-A3-M3 T306R, L180C, A223CT306R, L180C, A223C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 6161 Hy2-A3-M4Hy2-A3-M4 T306R, P6C, Y403CT306R, P6C, Y403C LNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 6262 Hy2-A3-M5Hy2-A3-M5 T306R, S36C, D426CT306R, S36C, D426C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYN 6363 Hy2-A4Hy2-A4 L307KL307K LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 6464 Hy2-A4-M1Hy2-A4-M1 L307K, P80C, A160CL307K, P80C, A160C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 6565 Hy2-A4-M2Hy2-A4-M2 L307K, G291C, Y362CL307K, G291C, Y362C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 6666 Hy2-A4-M3Hy2-A4-M3 L307K, L180C, A223CL307K, L180C, A223C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 6767 Hy2-A4-M4Hy2-A4-M4 L307K, P6C, Y403CL307K, P6C, Y403C LNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 6868 Hy2-A4-M5Hy2-A4-M5 L307K, S36C, D426CL307K, S36C, D426C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYN 6969 Hy2-A5Hy2-A5 T306AT306A LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 7070 Hy2-A5-M1Hy2-A5-M1 T306A, P80C, A160CT306A, P80C, A160C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 7171 Hy2-A5-M2Hy2-A5-M2 T306A, G291C, Y362CT306A, G291C, Y362C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 7272 Hy2-A5-M3Hy2-A5-M3 T306A, L180C, A223CT306A, L180C, A223C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 7373 Hy2-A5-M4Hy2-A5-M4 T306A, P6C, Y403CT306A, P6C, Y403C LNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNLNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTL SCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 7474 Hy2-A5-M5Hy2-A5-M5 T306A, S36C, D426CT306A, S36C, D426C LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYNLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCF NVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTL SCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYN 7575 Hy3-M1Hy3-M1 P80C, A160CP80C, A160C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 7676 Hy3-M2Hy3-M2 G291C, Y362CG291C, Y362C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKE KADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 7777 Hy3-M3Hy3-M3 L180C, A223CL180C, A223C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 7878 Hy3-M4Hy3-M4 P6C, Y403CP6C, Y403C FRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 7979 Hy3-M5Hy3-M5 S36C, D426CS36C, D426C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYN 8080 Hy3-A1Hy3-A1 Y57HY57H FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 8181 Hy3-A1-M1Hy3-A1-M1 Y57H, P80C, A160CY57H, P80C, A160C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 8282 Hy3-A1-M2Hy3-A1-M2 Y57H, G291C, Y362CY57H, G291C, Y362C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKE KADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 8383 Hy3-A1-M3Hy3-A1-M3 Y57H, L180C, A223CY57H, L180C, A223C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 8484 Hy3-A1-M4Hy3-A1-M4 Y57H, P6C, Y403CY57H, P6C, Y403C FRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 8585 Hy3-A1-M5Hy3-A1-M5 Y57H, S36C, D426CY57H, S36C, D426C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYN 8686 Hy3-A2Hy3-A2 W304HW304H FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKE KADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 8787 Hy3-A2-M1Hy3-A2-M1 W304H, P80C, A160CW304H, P80C, A160C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKE KADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 8888 Hy3-A2-M2Hy3-A2-M2 W304H, G291C, Y362CW304H, G291C, Y362C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 8989 Hy3-A2-M3Hy3-A2-M3 W304H, L180C, A223CW304H, L180C, A223C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKE KADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 9090 Hy3-A2-M4Hy3-A2-M4 W304H, P6C, Y403CW304H, P6C, Y403C FRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSKE KADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 9191 Hy3-A2-M5Hy3-A2-M5 W304H, S36C, D426CW304H, S36C, D426C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFHVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIHGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKE KADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYN 9292 Hy3-A3Hy3-A3 T306RT306R FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 9393 Hy3-A3-M1Hy3-A3-M1 T306R, P80C, A160CT306R, P80C, A160C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 9494 Hy3-A3-M2Hy3-A3-M2 T306R, G291C, Y362CT306R, G291C, Y362C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKE KADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 9595 Hy3-A3-M3Hy3-A3-M3 T306R, L180C, A223CT306R, L180C, A223C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 9696 Hy3-A3-M4Hy3-A3-M4 T306R, P6C, Y403CT306R, P6C, Y403C FRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 9797 Hy3-A3-M5Hy3-A3-M5 T306R, S36C, D426CT306R, S36C, D426C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGRLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYN 9898 Hy3-A4Hy3-A4 L307KL307K FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 9999 Hy3-A4-M1Hy3-A4-M1 L307K, P80C, A160CL307K, P80C, A160C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 100100 Hy3-A4-M2Hy3-A4-M2 L307K, G291C, Y362CL307K, G291C, Y362C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKE KADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 101101 Hy3-A4-M3Hy3-A4-M3 L307K, L180C, A223CL307K, L180C, A223C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 102102 Hy3-A4-M4Hy3-A4-M4 L307K, P6C, Y403CL307K, P6C, Y403C FRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCCSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 103103 Hy3-A4-M5Hy3-A4-M5 L307K, S36C, D426CL307K, S36C, D426C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTKSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYN 104104 Hy3-A5Hy3-A5 T306AT306A FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 105105 Hy3-A5-M1Hy3-A5-M1 T306A, P80C, A160CT306A, P80C, A160C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGICQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKCGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 106106 Hy3-A5-M2Hy3-A5-M2 T306A, G291C, Y362CT306A, G291C, Y362C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFCETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDCLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKE KADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 107107 Hy3-A5-M3Hy3-A5-M3 T306A, L180C, A223CT306A, L180C, A223C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHCWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTCLYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 108108 Hy3-A5-M4Hy3-A5-M4 T306A, P6C, Y403CT306A, P6C, Y403C FRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNFRACPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCCCSTLSC KEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYN 109109 Hy3-A5-M5Hy3-A5-M5 T306A, S36C, D426CT306A, S36C, D426C FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMCLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGALSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSC KEKADVKDTDAVDVCIACGVCIDAFLKPPMETEEPQIFYN

또한, 본 발명은 상기 본 발명에 따른 히알루로니다제 PH-20 변이체를 암호화(코딩)하는 폴리뉴클레오티드를 제공한다. Additionally, the present invention provides a polynucleotide encoding the hyaluronidase PH-20 variant according to the present invention.

본 발명의 폴리뉴클레오티드는 세포, 세포 용해물(lysate) 중에 존재하거나, 또는 부분적으로 정제된 형태 또는 실질적으로 순수한 형태로 존재할 수 있다. 폴리뉴클레오티드는 알칼리/SDS 처리, CsCl 밴드화(banding), 컬럼 크로마토그래피, 아가로스 겔 전기 영동 및 당업계에 잘 알려진 기타의 것을 포함하는 표준 기술에 의해 다른 세포 성분 또는 기타 오염 물질, 예를 들어, 다른 세포의 폴리뉴클레오티드 또는 단백질로부터 정제되어 나올 경우 '단리'되거나 '실질적으로 순수하게 된' 것이다. 상기 폴리뉴클레오티드는 본 발명의 PH-20 변이체를 암호화할 수 있는 한 폴리뉴클레오타이드의 염기 조합이 특별히 제한되지 않는다. 상기 폴리뉴클레오티드는 DNA, cDNA 및 RNA 서열을 모두 포함하여 단쇄 또는 이중쇄의 형태의 핵산 분자로서 제공될 수 있다.The polynucleotide of the present invention may be present in cells, cell lysates, or in partially purified or substantially pure form. Polynucleotides can be separated from other cellular components or other contaminants by standard techniques including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. , is ‘isolated’ or ‘substantially pure’ when it is purified from a polynucleotide or protein from another cell. The base combination of the polynucleotide is not particularly limited as long as it can encode the PH-20 variant of the present invention. The polynucleotide may be provided as a nucleic acid molecule in the form of a single strand or a double strand, including all DNA, cDNA, and RNA sequences.

본 발명의 PH-20 변이체를 암호화하는 핵산 서열을 이를 발현할 수 있는 벡터에 작동적으로 연결시켜 상기 본 발명의 PH-20 변이체를 제공할 수 있다. 따라서, 본 발명은 상기 폴리뉴클레오타이드를 포함하는 발현 벡터 또는 재조합 벡터를 제공한다. The PH-20 variant of the present invention can be provided by operably linking the nucleic acid sequence encoding the PH-20 variant of the present invention to a vector capable of expressing it. Accordingly, the present invention provides an expression vector or recombinant vector containing the above polynucleotide.

본 발명에서 용어 '발현 벡터' 또는 '재조합 벡터'란 적당한 숙주세포에서 목적 단백질 또는 목적 RNA을 발현할 수 있는 벡터로서, 유전자 삽입물이 발현되도록 작동 가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 제작물을 말한다.In the present invention, the term 'expression vector' or 'recombinant vector' refers to a vector capable of expressing a target protein or target RNA in a suitable host cell, and refers to a gene construct containing essential regulatory elements operably linked to express the gene insert. .

상기 용어 '작동 가능하게 연결된(operably linked)'는 일반적 기능을 수행하도록 핵산 발현 조절 서열과 목적하는 단백질 또는 RNA를 코딩하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 것을 말한다. 예를 들어 프로모터와 단백질 또는 RNA를 코딩하는 핵산 서열이 작동 가능하게 연결되어 코딩하는 핵산 서열의 발현에 영향을 미칠 수 있다. 재조합 벡터와의 작동적 연결은 당해 기술 분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용한다.The term 'operably linked' refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect expression of the encoding nucleic acid sequence. Operational linkage with a recombinant vector can be made using genetic recombination techniques well known in the art, and site-specific DNA cutting and ligation can be done using enzymes generally known in the art.

하나의 실시 양태에서, 상기 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오파아지 벡터 및 바이러스 벡터 등을 포함하나 이에 제한되지 않는다. 적합한 발현 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 등과 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. 또한 발현 벡터는 벡터를 함유하는 숙주 세포를 선택하기 위한 선택 마커를 포함하고, 복제 가능한 발현 벡터인 경우 복제 기원을 포함할 수 있다.In one embodiment, the vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc. A suitable expression vector includes expression control elements such as a promoter, operator, start codon, stop codon, polyadenylation signal, and enhancer, as well as a signal sequence or leader sequence for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The expression vector may also include a selection marker for selecting host cells containing the vector, and, if it is a replicable expression vector, may include an origin of replication.

시그널 서열에는 숙주가 에스케리치아 속(Escherichia sp.) 균인 경우에는 PhoA 시그널 서열, OmpA 시그널 서열 등이, 숙주가 바실러스속 균인 경우에는 α-아밀라아제 시그널 서열, 서브틸리신 시그널 서열 등이, 숙주가 효모인 경우에는 MFα 시그널 서열, SUC2 시그널 서열 등이, 숙주가 동물세포인 경우에는 인슐린 시그널서열, α-인터페론 시그널 서열, 항체 분자 시그널 서열 등을 이용할 수 있으나, 이에 제한되지 않는다.The signal sequence includes the PhoA signal sequence and OmpA signal sequence when the host is a genus Escherichia ( Escherichia sp.), and the α-amylase signal sequence and subtilisin signal sequence when the host is a bacterium of the genus Bacillus. In the case of yeast, the MFα signal sequence, SUC2 signal sequence, etc. can be used, and if the host is an animal cell, the insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used, but are not limited thereto.

또한, 본 발명은 상기 발현 벡터를 포함하는 숙주 세포주를 제공한다. 즉, 본 발명은 상기 발현 벡터(재조합 벡터)로 형질전환된 형질전환체(숙주 세포)를 제공한다.Additionally, the present invention provides a host cell line containing the expression vector. That is, the present invention provides a transformant (host cell) transformed with the expression vector (recombinant vector).

상기 형질전환은 핵산을 유기체, 세포, 조직 또는 기관에 도입할 수 있는 것으로 공지된 것이라면 어떤 방법이라도 사용 가능하며, 당 분야에서 공지된 바와 같이 숙주 세포에 따라 적합한 표준 기술을 선택하여 수행할 수 있다. 이런 방법에는 미세사출법(microprojectile bombardment), 전기충격유전자전달법(electroporation), 원형질 융합, 인산 칼슘(CaPO₄) 침전, 염화 칼슘(CaCl₂) 침전, 실리콘 카바이드 섬유 이용한 교반, 아그로 박테리아 매개된 형질전환, PEG-매개 융합법(PEG-mediated fusion), 미세주입법(microinjection), 리포좀 매개법(liposome-mediated method), 덱스트란 설페이트, 리포펙타민, 열충격법 등이 포함되나, 이로 제한되지 않는다.The transformation can be performed by any method known to be capable of introducing nucleic acids into an organism, cell, tissue or organ, and can be performed by selecting an appropriate standard technique depending on the host cell, as known in the art. . These methods include microprojectile bombardment, electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation using silicon carbide fibers, and agrobacteria-mediated transformation. Conversion, PEG-mediated fusion, microinjection, liposome-mediated method, dextran sulfate, lipofectamine, heat shock method, etc. are included, but are not limited thereto.

상기 용어 '형질전환체'는 '숙주 세포' 등과 호환성 있게 사용될 수 있으며, 임의의 수단(예: 전기충격법, 칼슘 포스파타제 침전법, 미세주입법, 형질전환법, 바이러스 감염 등)에 의해 세포 내로 도입된 이종성 DNA를 포함하는 원핵 또는 진핵 세포를 의미한다.The term 'transformant' can be used interchangeably with 'host cell', etc., and can be introduced into a cell by any means (e.g. electric shock method, calcium phosphatase precipitation method, microinjection method, transformation method, virus infection, etc.). refers to a prokaryotic or eukaryotic cell containing heterologous DNA.

본 발명에서 상기 형질전환체는 클로닝 분야에서 통상적으로 사용되는 모든 종류의 단세포 유기체, 예컨대 각종 박테리아 (예컨대, Clostridia 속, 대장균, 등) 등의 원핵세포 미생물, 효모 등의 하등 진핵세포 미생물과 곤충 세포, 식물 세포, 포유동물 등을 포함하는 고등 진핵생물 유래의 세포를 숙주세포로 사용할 수 있으며, 이에 제한되지 않는다. 숙주세포에 따라서 단백질의 발현량과 수식 등이 다르게 나타나므로 당업자가 목적하는 바에 가장 적합한 숙주세포를 선택하여 사용할 수 있다. 본 발명의 상기 형질전환체는 바람직하게 형질전환 미생물을 의미하는 것일 수 있다. 구체적으로, 이에 제한되지 않으나, 예를 들어 숙주세포로는 에스케리치아 콜라이(대장균, Escherichia coli), 바실러스 서브틸리스(Bacillus subtilis), 스트렙토마이세스(Streptomyces), 슈도모나스(Pseudomonas), 프로테우스 미라빌리스(Proteus mirabilis) 또는 스타필로코쿠스(Staphylococcus)와 같은 원핵 숙주 세포일 수 있다. 또한, 진균(예를 들어, 아스페르길러스(Aspergillus)), 효모(예를 들어, 피치아 파스토리스(Pichia pastoris), 사카로마이세스 세르비지애(Saccharomyces cerevisiae), 쉬조사카로미세스(Schizosaccharomyces), 뉴로스포라크라사(Neurospora crassa))등과 같은 하등 진핵 세포, 곤충 세포, 식물 세포, 포유동물 등을 포함하는 고등 진핵생물 유래의 세포를 숙주세포로 사용할 수 있다. 바람직하게는, 원숭이 신장 세포7(COS7: monkey kidney cells), NSO 세포, SP2/0, 차이니즈 햄스터 난소(CHO: Chinese hamster ovary) 세포, W138, 어린 햄스터 신장(BHK: baby hamster kidney)세포, MDCK, 골수종 세포주, HuT 78 세포 및 HEK293 세포 등이 이용 가능하며, 특히 바람직하게는 CHO 세포가 사용될 수 있으나, 이에 제한되지 않는다. In the present invention, the transformant includes all types of single-celled organisms commonly used in the cloning field, such as prokaryotic microorganisms such as various bacteria (e.g., Clostridia genus, Escherichia coli, etc.), lower eukaryotic microorganisms such as yeast, and insect cells. , cells derived from higher eukaryotes, including plant cells, mammals, etc., can be used as host cells, but are not limited thereto. Since the expression level and modification of proteins varies depending on the host cell, a person skilled in the art can select and use the host cell most suitable for the purpose. The transformant of the present invention may preferably refer to a transformed microorganism. Specifically, but not limited thereto, host cells include, for example, Escherichia coli, Bacillus subtilis , Streptomyces , Pseudomonas , and Proteus mirabili. It may be a prokaryotic host cell such as Proteus mirabilis or Staphylococcus . Additionally, fungi (e.g., Aspergillus ), yeast (e.g., Pichia pastoris , Saccharomyces cerevisiae ), Schizosaccharomyces ), Neurospora crassa ( Neurospora crassa ), etc. Cells derived from lower eukaryotes, including insect cells, plant cells, mammals, etc., can be used as host cells. Preferably, monkey kidney cells (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK. , myeloma cell lines, HuT 78 cells, and HEK293 cells can be used, and CHO cells are particularly preferably used, but are not limited thereto.

또한, 본 발명은 상기 숙주 세포주를 배양하는 단계를 포함하는 히알루로니다제 PH-20 변이체의 제조 방법을 제공한다.Additionally, the present invention provides a method for producing a hyaluronidase PH-20 variant comprising culturing the host cell line.

상기 PH-20 변이체 또는 이의 절편을 발현할 수 있는 재조합 발현 벡터가 숙주 세포 내로 도입될 경우, PH-20 변이체는 숙주 세포에서 발현되기에 충분한 기간 동안, 더 바람직하게는 숙주 세포가 배양되는 배양 배지 내로 PH-20 변이체가 분비되게 하기에 충분한 기간 동안 숙주 세포를 배양함으로써 제조될 수 있다.When a recombinant expression vector capable of expressing the PH-20 variant or a fragment thereof is introduced into a host cell, the PH-20 variant is expressed for a period sufficient to be expressed in the host cell, more preferably in a culture medium in which the host cell is cultured. It can be prepared by culturing the host cell for a period sufficient to cause secretion of the PH-20 variant into the cell.

상기 숙주 세포를 배양하기 위한 배지 조성과 배양 조건, 배양 시간 등은 당업계에서 통상적으로 사용되는 방법에 따라 적절하게 선택할 수 있다. 숙주 세포에서 생산되는 PH-20 변이체는 세포의 세포질 내에 축적되거나, 적절한 신호 서열에 의하여 세포 외부 또는 배양 배지로 분비되거나, 페리플라즘 등으로 표적화 되도록 할 수 있다. 또한, 본 발명에 따른 PH-20 변이체가 그 효소 활성을 유지하도록 당업계에 공지되어 있는 방법을 이용하여 단백질을 리폴딩(refolding)시키고, 기능성 구조(conformation)를 갖도록 하는 것이 바람직하다.The medium composition, culture conditions, culture time, etc. for culturing the host cells can be appropriately selected according to methods commonly used in the art. PH-20 variants produced in host cells can accumulate in the cell cytoplasm, be secreted outside the cell or into the culture medium by an appropriate signal sequence, or be targeted to periplasm, etc. In addition, it is desirable to refold the protein using a method known in the art so that the PH-20 variant according to the present invention maintains its enzymatic activity and have a functional conformation.

경우에 따라서, 발현된 PH-20 변이체는 숙주 세포로부터 분리하여 균일하도록 정제할 수 있다. 상기 PH-20 변이체의 분리 또는 정제는 통상의 단백질에서 사용되고 있는 분리, 정제 방법, 예를 들어, 크로마토그래피에 의해 수행될 수 있다. 상기 크로마토그래피는 예를 들어, 친화성 크로마토그래피, 이온교환 크로마토그래피, 또는 소수성 크로마토그래피에서 선택된 하나 이상의 조합일 수 있으나, 이에 제한되는 것은 아니다. 상기 크로마토그래피 이외에 추가로 여과, 초여과, 염석, 투석 등을 조합하여 사용할 수 있다. In some cases, the expressed PH-20 variant can be isolated from the host cell and purified to uniformity. Separation or purification of the PH-20 variant can be performed by separation and purification methods used for conventional proteins, for example, chromatography. The chromatography may be, for example, a combination of one or more selected from affinity chromatography, ion exchange chromatography, or hydrophobic chromatography, but is not limited thereto. In addition to the above chromatography, filtration, ultrafiltration, salting out, dialysis, etc. can be used in combination.

또한, 본 발명은 a) 약물; 및 b) 상기 히알루로니다제 PH-20 변이체;를 포함하는 것을 특징으로 하는 질환의 예방 또는 치료용 약학적 조성물을 제공한다.Additionally, the present invention relates to a) a drug; and b) the hyaluronidase PH-20 variant. It provides a pharmaceutical composition for preventing or treating diseases, comprising:

본 발명에 따른 약학적 조성물에 있어, 상기 약물은 단백질 의약품(protein drugs), 항체 의약품, 소분자 화합물(small molecules), 앱타머(aptamer), RNAi, antisence, CAR-T 세포 (chimeric antigen receptor T cell) 또는 CAR-NK 세포 (CAR natural killer cell) 등의 세포 치료제 등이 포함될 수 있으나, 이에 한정되는 것은 아니며, 현재 상용화되어 사용되고 있는 약물 뿐만 아니라, 임상 및 개발이 진행 중에 있는 약물이 모두 사용 가능하다. 상기 약물로는 바람직하게는 단백질 의약품이나 항체 의약품이 사용될 수 있다. In the pharmaceutical composition according to the present invention, the drug is protein drugs, antibody drugs, small molecules, aptamers, RNAi, antisence, CAR-T cells (chimeric antigen receptor T cell) ) or cell therapy such as CAR-NK cells (CAR natural killer cell), but are not limited to this, and not only drugs that are currently commercialized and used, but also drugs that are in clinical trials and development can be used. . Preferably, protein drugs or antibody drugs can be used as the drug.

본 발명에 따른 약학적 조성물에 있어, 상기 '단백질 의약품'은 아미노산으로 구성되어 단백질의 활성을 통해 질병의 예방 또는 치료 효과를 나타내는 약물로서, 항체 의약품 이외의 단백질로 이루어진 의약품을 의미하며, 사이토카인(cytokines), 치료용 효소(therapeutic enzyme), 호르몬(hormone), 수용성 수용체(soluble receptor) 및 이의 융합 단백질, 인슐린(insulin) 또는 이의 유사체, BMP(bone morphogenetic protein), EPO (erythropoietin) 및 혈장 유래 단백질(serum derived protein) 등으로 구성된 군에서 선택될 수 있으나, 이에 한정되는 것은 아니다. In the pharmaceutical composition according to the present invention, the 'protein drug' refers to a drug composed of amino acids and showing a disease prevention or treatment effect through the activity of protein, and refers to a drug composed of proteins other than antibody drugs, and cytokines. (cytokines), therapeutic enzymes, hormones, soluble receptors and their fusion proteins, insulin or its analogs, BMP (bone morphogenetic protein), EPO (erythropoietin) and plasma derived It may be selected from the group consisting of proteins (serum derived proteins), etc., but is not limited thereto.

상기 '사이토카인'은 인터페론(interferon), 인터루킨(interleukin), CSF (colony stimulating factor), TNF (tumor necrosis factor) 및 TGF (tissue growth factor) 등으로 구성된 군에서 선택될 수 있지만 이에 한정되는 것은 아니며, 상기 '치료용 효소'는 베타-글루코세레브로시다제(β-glucocerebrosidase) 및 아갈시다제 베타(agalsidase β) 등이 예시될 수 있지만 이에 한정되는 것은 아니다. The 'cytokine' may be selected from the group consisting of interferon, interleukin, CSF (colony stimulating factor), TNF (tumor necrosis factor), and TGF (tissue growth factor), but is not limited thereto. , The 'therapeutic enzyme' may include, but is not limited to, beta-glucocerebrosidase and agalsidase beta.

상기 '수용성 수용체'는 수용체의 세포 외 도메인(extracellular domain)을 의미하고, 이의 융합 단백질은 상기 수용성 수용체에 항체의 Fc 영역 등이 융합된 단백질을 의미한다. 상기 수용성 수용체는 질병과 관련된 리간드가 결합하는 수용체의 수용성 형태로서, TNF-α 수용성 수용체에 Fc 영역이 융합된 형태(예를 들어, 에타너셉트(etanercept)라는 성분명의 제품 및 이와 유사한 형태) 및 VEGF 수용성 수용체에 Fc 영역이 융합된 형태(예를 들어, 아플리버셉트(Aflibercept)라는 성분명의 제품 및 이와 유사한 형태), CTLA-4에 Fc 영역이 융합된 형태(예를 들어, 아바타셉트(Abatacept) 또는 벨라다셉트(Belatacept)라는 성분명의 제품 및 이와 유사한 형태), 인터루킨1 수용성 수용체에 Fc 영역이 융합된 형태(예를 들어, 릴로나셉트(Rilonacept)라는 성분명의 제품 및 이와 유사한 형태), LFA3 수용성 수용체에 Fc 영역이 융합된 형태(예를 들어, 알레파셉트(Alefacept) 라는 성분명의 제품 및 이와 유사한 형태) 등이 예시될 수 있지만, 이에 한정되는 것은 아니다. The 'soluble receptor' refers to the extracellular domain of the receptor, and its fusion protein refers to a protein in which the Fc region of an antibody, etc. is fused to the soluble receptor. The water-soluble receptor is a water-soluble form of the receptor to which the disease-related ligand binds, and is a form in which the Fc region is fused to the TNF-α water-soluble receptor (for example, a product with the ingredient name etanercept and similar forms) and VEGF water-soluble receptor. A form in which the Fc region is fused to a receptor (e.g., a product with the ingredient name Aflibercept and similar forms), a form in which the Fc region is fused to CTLA-4 (e.g., Abatacept or Bellacept) Dacept (Belatacept and similar forms), a form in which the Fc region is fused to the interleukin 1 soluble receptor (e.g., a product with the ingredient name Rilonacept and similar forms), LFA3 soluble receptor Examples include a form in which the Fc region is fused (for example, a product with the ingredient name Alefacept and similar forms), but it is not limited thereto.

상기 '호르몬'은 호르몬 결핍 등으로 인해 발생하는 질환의 치료 또는 예방을 위해 체외에서 주입하는 호르몬 또는 이의 유사체를 의미하며, 사람 성장호르몬(human growth hormone), 에스트로겐(estrogen), 프로게스테론(progesterone) 등이 예시될 수 있지만 이에 한정되는 것은 아니다. 상기 '혈장 유래 단백질'은 혈장에 존재하는 단백질로, 혈장에서 추출한 것과 재조합으로 생산된 것들 모두를 의미하며, 피브리노겐(fibrinogen), 본 빌레블란트 인자(von Willebrand Factor), 알부민(albumin), 트롬빈(thrombin), FII (Factor II), FV (Factor V), FVII (Factor VII), FVIII (Factor VIII), FIX (Factor IX), FX (Factor X) 및 FXI (Factor XI) 등이 예시될 수 있지만 이에 한정되는 것은 아니다.The term 'hormone' refers to a hormone or its analogue injected from outside the body for the treatment or prevention of diseases caused by hormone deficiency, etc., such as human growth hormone, estrogen, progesterone, etc. This may be exemplified, but is not limited to this. The 'plasma-derived protein' refers to a protein present in plasma, both extracted from plasma and recombinantly produced, including fibrinogen, von Willebrand Factor, albumin, and thrombin. (thrombin), FII (Factor II), FV (Factor V), FVII (Factor VII), FVIII (Factor VIII), FIX (Factor IX), FX (Factor However, it is not limited to this.

본 발명에 따른 약학적 조성물에 있어, 상기 '항체 의약품'은 단클론 항체 의약품(monoclonal antibody drug) 또는 다클론 항체 의약품(polyclonal antibody drug)일 수 있다. In the pharmaceutical composition according to the present invention, the 'antibody drug' may be a monoclonal antibody drug or a polyclonal antibody drug.

상기 '단클론 항체 의약품'은 특정 질병과 관련된 항원에 특이적으로 결합할 수 있는 단클론 항체 및 단클론 항체 단편을 포함한 단백질을 의미한다. 단클론 항체에는 이중 항체도 포함되며, 단클론 항체 또는 이의 단편을 포함하는 단백질은 ADC (antibody-drug conjugate)를 포함하는 의미로 사용된다. 특정 질병과 관련된 항원은 4-1BB, 인테그린(integrin), 아밀로이드 베타(amyloid beta), 안지오포에틴(안지오포에틴 1 또는 2), 안지오포에틴 유사물질3, B세포 활성인자(B-cell activating factor, BAFF), B7-H3, 보체5 (complement 5), CCR4, CD3, CD4, CD6, CD11a, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD62, CD79b, CD80, CGRP, 클라우딘18 (Claudin-18), 보체요소 D (complement factor D), CTLA4, DLL3, EGF 수용체, 혈우병인자, Fc 수용체, FGF23, 폴레이트 (folate) 수용체, GD2, GM-CSF, HER2, HER3, 인터페론 수용체, 인터페론 감마, IgE, IGF-1 수용체, 인터루킨1, 인터루킨2 수용체, 인터루킨4 수용체, 인터루킨5, 인터루킨5 수용체, 인터루킨6, 인터루킨6 수용체, 인터루킨7, 인터루킨12/23, 인터루킨13, 인터루킨17A, 인터루킨17수용체A, 인터루킨31 수용체, 인터루킨36 수용체, LAG3, LFA3, NGF, PVSK9, PD-1, PD-L1, RANK-L, SLAMF7, 조직인자 (tissue factor), TNF, VEGF, VEGF 수용체 및 vWF (von Willebrand Factor)이며, 이에 한정되는 것은 아니다. 이하는 상기 특정 질병과 관련된 항원에 대한 단클론 항체 및 단클론 항체 단편을 포함하는 단백질이다;The ‘monoclonal antibody drug’ refers to a protein containing monoclonal antibodies and monoclonal antibody fragments that can specifically bind to antigens associated with specific diseases. Monoclonal antibodies also include double antibodies, and proteins containing monoclonal antibodies or fragments thereof are used to include ADC (antibody-drug conjugate). Antigens associated with specific diseases include 4-1BB, integrin, amyloid beta, angiopoietin (angiopoietin 1 or 2), angiopoietin-like substance 3, and B-cell activating factor. factor, BAFF), B7-H3, complement 5, CCR4, CD3, CD4, CD6, CD11a, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD62, CD79b, CD80, CGRP, Claudin 18 (Claudin-18), complement factor D, CTLA4, DLL3, EGF receptor, hemophilia factor, Fc receptor, FGF23, folate receptor, GD2, GM-CSF, HER2, HER3, interferon receptor, Interferon gamma, IgE, IGF-1 receptor, interleukin 1, interleukin 2 receptor, interleukin 4 receptor, interleukin 5, interleukin 5 receptor, interleukin 6, interleukin 6 receptor, interleukin 7, interleukin 12/23, interleukin 13, interleukin 17A, interleukin 17Receptor A, interleukin 31 receptor, interleukin 36 receptor, LAG3, LFA3, NGF, PVSK9, PD-1, PD-L1, RANK-L, SLAMF7, tissue factor, TNF, VEGF, VEGF receptor and vWF ( von Willebrand Factor), and is not limited to this. The following are proteins containing monoclonal antibodies and monoclonal antibody fragments against antigens associated with the above-mentioned specific diseases;

4-1BB에 대한 항체로 유토밀루맙(Utomilumab);Utomilumab as an antibody against 4-1BB;

인테그린에 대한 항체로 나탈리주맙(Natalizumab), 에트롤리주맙(Etrolizumab), 베돌리주맙(Vedolizumab), 비마그루맙(Bimagrumab);Antibodies against integrins include Natalizumab, Etrolizumab, Vedolizumab, and Bimagrumab;

아밀로이드 베타에 대한 항체로 바피네우주맙(Bapineuzumab), 크레네주맙(Crenezumab), 솔라네주맙(Solanezumab), 아두카누맙(Aducanumab), 간테네루맙(Gantenerumab);Antibodies against amyloid beta include Bapineuzumab, Crenezumab, Solanezumab, Aducanumab, and Gantenerumab;

안지오포에틴에 대한 항체로 안지오포에틴 1과 2에 대한 AMG 780, 안지오포에틴 2에 대한 MEDI 3617, 네스바쿠맙(Nesvacumab)과 안지오포에틴 2와 VEGF의 이중항체인 바누씨주맙(Vanucizumab);Antibodies to angiopoietin include AMG 780 against angiopoietin 1 and 2, MEDI 3617 against angiopoietin 2, Nesvacumab, and Vanucizumab, a double antibody against angiopoietin 2 and VEGF. ;

안지오포에틴 유사물질3에 대한 항체로 에비나쿠맙(Evinacumab);Evinacumab, an antibody against angiopoietin-like substance 3;

B세포 활성인자(B-cell activating factor, BAFF)에 대한 항체로 타발루맙(Tabalumab), 라나루맙(Lanalumab), 베리무맙(Belimumab);Antibodies against B-cell activating factor (BAFF) include Tabalumab, Lanalumab, and Belimumab;

B7-H3에 대한 항체로 옴부르타맙(omburtamab);Omburtamab, an antibody against B7-H3;

보체5(complement 5)에 대한 항체로 라불리주맙(Ravulizumab), 에쿨리주맙(Eculizumab);Antibodies against complement 5 include Ravulizumab and Eculizumab;

CCR4에 대한 항체로 모가물리주맙(Mogamulizumab);Mogamulizumab as an antibody against CCR4;

CD3에 대한 항체로 오텔릭시주맙(Otelixizumab), 테플리주맙(Teplizumab), 무로모납(Muromonab)과 GP100과 CD3에 대한 이중 항체인 테벤타프스프(Tebentafusp), CD19와 CD3에 대한 이중항체인 블리나투모맙(Blinatumomab); CD20과 CD3에 대한 이중 항체인 REGN1979;Otelixizumab, Teplizumab, and Muromonab are antibodies against CD3, and Tebentafusp, a double antibody against GP100 and CD3, is a double antibody against CD19 and CD3. Blinatumomab; REGN1979, a double antibody against CD20 and CD3;

CD4에 대한 항체로 이발리주맙(Ibalizumab), 자놀리무맙(Zanolimumab);Antibodies against CD4 include Ibalizumab and Zanolimumab;

CD6에 대한 항체로 이톨리주맙(Itolizumab);Itolizumab, an antibody against CD6;

CD11a에 대한 항체로 에팔리주맙(Efalizumab);Efalizumab, an antibody against CD11a;

CD19에 대한 항체로 이네빌리주맙(Inebilizumab), 타파시타맙(Tafasitamab) 과 ADC인 론카스툭시맙 테시린(Loncastuximab tesirine);Antibodies against CD19 include Inebilizumab and Tafasitamab and Loncastuximab tesirine, an ADC;

CD20에 대한 항체로 오크렐리주맙(Ocrelizumab), 우블리툭시맙(Ublituximab), 오비누투주맙(Obinutuzumab), 오파투무맙(Ofatumumab), 리툭시맙(Rituximab), 토시투모맙(Tositumomab)과 ADC인 이브리투모맙 티욱세탄(Ibritumomab tiuxetan);Antibodies against CD20 include Ocrelizumab, Ublituximab, Obinutuzumab, Ofatumumab, Rituximab, and Tositumomab. Ibritumomab tiuxetan, an ADC;

CD22에 대한 항체로 에프라투주맙(Epratuzumab)과 ADC인 이노투주맙 오조가마이신(Inotuzumab ozogamicin), 목세투모맙 파수도톡스(Moxetumomab pasudotox);Epratuzumab, an antibody against CD22, and ADCs Inotuzumab ozogamicin and Moxetumomab pasudotox;

CD30에 대한 ADC는 브렌툭시맙 베도틴(Brentuximab vedotin);ADCs for CD30 include brentuximab vedotin;

CD33에 대한 ADC는 바다스툭시맙 탈리닌(Vadastuximab talirine), 젬투주맙 오조가마이신(Gemtuzumab ozogamicin);ADCs for CD33 include Vadastuximab talirine, Gemtuzumab ozogamicin;

CD38에 대한 항체로 다라투무맙(Daratumumab), 이사툭시맙(Isatuximab);Antibodies against CD38 include Daratumumab and Isatuximab;

CD52에 대한 항체로 알렘투주맙(Alemtuxumab);Alemtuxumab, an antibody against CD52;

CD62에 대한 항체로 크리잔리주맙(Crizanlizumab);Crizanlizumab, an antibody against CD62;

CD79b에 대한 ADC는 폴라투주맙 베도틴(Polatuzumab vedotin);ADCs for CD79b include Polatuzumab vedotin;

CD80에 대한 항체로 갈릭시맙(Galiximab);Galiximab, an antibody against CD80;

CGRP에 대한 항체로 žƒ티네주맙(Eptinezumab), 프레마네주맙(Fremanezumab), 갈카네주맙(Galcanezumab), 에레누맙(Erenumab);Antibodies to CGRP include Eptinezumab, Fremanezumab, Galcanezumab, and Erenumab;

클라우딘18(Claudin-18) 에 대한 항체로 졸베툭시맙(Zolbetuximab);Zolbetuximab as an antibody against Claudin-18;

보체요소 D(complement factor D)에 대한 항체로 람팔리주맙(Lampalizumab);Lampalizumab, an antibody against complement factor D;

CTLA4에 대한 항체로 트레멜리무맙(Tremelimumab), 잘리프렐리맙(Zalifrelimab), 이플리무맙(Ipilimumab);Antibodies against CTLA4 include Tremelimumab, Zaliprelimab, and Ipilimumab;

DLL3에 대한 ADC는 로발피투주맙 테시린(Rovalpituzumab tesirine);ADCs for DLL3 include Rovalpituzumab tesirine;

EGF 수용체에 대한 항체로 세툭시맙(Cetuximab), 데파툭시주맙(Depatuxizumab), 잘루투무맙(Zalutumumab), 네시투무맙(Necitumumab), 파니투무맙(Panitumumab);Antibodies to the EGF receptor include Cetuximab, Depatuxizumab, Zalutumumab, Necitumumab, and Panitumumab;

혈우병 인자인 coagulation factor IX 및 Factor X에 대한 이중 항체로 에미시주맙(Emicizumab);Emicizumab, a dual antibody against the hemophilia factors coagulation factor IX and factor X;

Fc 수용체에 대한 항체로 니포칼리맙(Nipocalimab), 로잔놀릭시주맙(Rozanolixizumab);Antibodies against Fc receptors include Nipocalimab and Rozanolixizumab;

FGF23에 대한 항체로 부로스맙(Burosumab);Burosumab, an antibody against FGF23;

폴레이트(folate) 수용체에 대한 항체로 팔레투주맙(Farletuzumab)과 ADC인 미르베툭시맙 소랍탄신(Mirvetuximab soravtansine);Farletuzumab, an antibody against the folate receptor, and Mirvetuximab soravtansine, an ADC;

GD2에 대한 항체로 디누툭시맙(Dinutuximab), 낙시타맙(Naxitamab);Antibodies against GD2 include dinutuximab and naxitamab;

GM-CSF에 대한 항체로 오틸리맙(Otilimab);Otilimab, an antibody against GM-CSF;

HER2에 대한 항체로 마르게툭시맙(Margetuximab), 페르투주맙(Pertuzumab), 트라스투주맙(Trastuzumab)과 ADC는 트라수투주맙 데룩테칸(Trastuzumab deruxtecan), 트라수투주맙 엠탄신(Trastuzumab emtansine), 트라수투주맙 두오카르마진(Trastuzumab duocarmazine);Antibodies against HER2 include Margetuximab, Pertuzumab, and Trastuzumab, and ADCs include Trastuzumab deruxtecan, Trastuzumab emtansine, and Trastuzumab. Trastuzumab duocarmazine;

HER3에 대한 항체로 파트리투맙(Patritumab);Patritumab, an antibody against HER3;

인터페론 수용체에 대한 항체로 아니프롤루맙(Anifrolumab);Anifrolumab, an antibody against the interferon receptor;

인터페론 감마에 대한 항체로 에마팔루맙(Emapalumab);Emapalumab, an antibody against interferon gamma;

IgE에 대한 항체로 리겔리주맙(Ligelizumab), 오말리주맙(Omalizumab);Antibodies to IgE include Ligelizumab and Omalizumab;

IGF-1 수용체에 대한 항체로 달로투주맙(Dalotuzumab), 피기투무맙(Figitumumab), 테프로투무맙(Teprotumumab);Antibodies to the IGF-1 receptor include Dalotuzumab, Figitumumab, and Teprotumumab;

인터루킨1에 대한 항체로 게보키주맙(Gebokizumab), 카나키누맙(Canakinumab);Antibodies against interleukin 1 include Gebokizumab and Canakinumab;

인터루킨2 수용체에 대한 항체로 다클리주맙(Daclizumab), 바실릭시맙(Basiliximab);Antibodies to the interleukin 2 receptor include Daclizumab and Basiliximab;

인터루킨4 수용체에 대한 항체로 두필루맙(Dupilumab);Dupilumab, an antibody against the interleukin 4 receptor;

인터루킨5에 대한 항체로 메폴리주맙(Mepolizumab), 레슬리주맙(Reslizumab);Antibodies against interleukin 5 include Mepolizumab and Reslizumab;

인터루킨5 수용체에 대한 항체로 벤랄리주맙(Benralizumab);Benralizumab, an antibody against the interleukin 5 receptor;

인터루킨6에 대한 항체로 클라자키주맙(Clazakizumab), 올로키주맙(Olokizumab), 시루쿠맙(Sirukumab), 실툭시맙(Siltuximab);Antibodies to interleukin 6 include Clazakizumab, Olokizumab, Sirukumab, and Siltuximab;

인터루킨6 수용체에 대한 항체로 사릴루맙(Sarilumab), 사트랄리주맙(Satralizumab), 토실리주맙(Tocilizumab), REGN88;Antibodies to the interleukin 6 receptor include Sarilumab, Satralizumab, Tocilizumab, and REGN88;

인터루킨7에 대한 항체로 세쿠키누맙(Secukinumab);Secukinumab as an antibody against interleukin 7;

인터루킨12/23에 대한 항체로 우스테키누맙(Ustekinumab), 브리아키누맙(Briakinumab);Antibodies against interleukin 12/23 include Ustekinumab and Briakinumab;

인터루킨13에 대한 항체로 레브리키주맙(Lebrikizumab), 트랄로키누맙(Tralokinumab);Antibodies against interleukin 13 include Lebrikizumab and Tralokinumab;

인터루킨17A에 대한 항체로 익세키주맙(Ixekizumab), 비메키주맙(Bimekizumab);Antibodies against interleukin 17A include Ixekizumab and Bimekizumab;

인터루킨17 수용체A에 대한 항체로 브로달루맙(Brodalumab);Brodalumab, an antibody against interleukin 17 receptor A;

인터루킨23에 대한 항체로 브라지쿠맙(Brazikumab), 구셀쿠맙(Guselkumab), 리산키주맙(Risankizumab), 틸드라키주맙(Tildrakizumab), 미리키주맙(Mirikizumab);Antibodies to interleukin 23 include Brazikumab, Guselkumab, Risankizumab, Tildrakizumab, and Mirikizumab;

인터루킨31 수용체에 대한 항체로 네몰리주맙(Nemolizumab);Nemolizumab, an antibody against the interleukin 31 receptor;

인터루킨36 수용체에 대한 항체로 스페솔리맙(Spesolimab);Spesolimab, an antibody against the interleukin 36 receptor;

LAG3에 대한 항체로 렐라틀리맙(Relatlimab);Relatlimab, an antibody against LAG3;

NASP2에 대한 항체로 나르소플리맙(Narsoplimab);Narsoplimab, an antibody against NASP2;

NGF에 대한 항체로 파시누맙(Fasinumab), 타네주맙(Tanezumab);Antibodies to NGF include Fasinumab and Tanezumab;

PVSK9에 대한 항체로 알리로쿠맙(Alirocumab), 에볼로쿠맙(Evolocumab), 보코시주맙(Bococizumab);Antibodies against PVSK9 include Alirocumab, Evolocumab, and Bococizumab;

PD-1에 대한 항체로 람브롤리주맙(Lambrolizumab), 발스틸리맙(Balstilimab), 캄렐리주맙(Camrelizumab), 쎄미플리맙(Cemiplimab), 도스탈리맙(Dostarlimab), 프롤골리맙(Prolgolimab), 신틸리맙(Sintilimab), 스파르탈리주맙(Spartalizumab), 티슬레리주맙(Tislelizumab), 펨브롤리주맙(Pembrolizumab), 니볼루맙(Nivolumab);Antibodies to PD-1 include Lambrolizumab, Balstilimab, Camrelizumab, Cemiplimab, Dostarlimab, Prolgolimab, and renal Sintilimab, Spartalizumab, Tislelizumab, Pembrolizumab, Nivolumab;

PD-L1에 대한 항체로 아테졸리주맙(Atezolizumab), 아벨루맙(Avelumab), 엔바폴리맙(Envafolimab), 둘발루맙(Durvalumab)과 TGF beta와 PD-L1의 이중 항체인 빈트라푸스프 알파(Bintrafusp alpha);Antibodies against PD-L1 include Atezolizumab, Avelumab, Envafolimab, and Durvalumab, and bintrafusp alfa (a double antibody against TGF beta and PD-L1). Bintrafusp alpha);

RANK-L에 대한 항체로 데노수맙(Denosumab);Denosumab, an antibody against RANK-L;

SLAMF7에 대한 항체로 엘로투주맙(Elotuzumab);Elotuzumab as an antibody against SLAMF7;

조직인자(Tissue factor)에 대한 항체로 콘시주맙(Concizumab), 말스타시맙(Marstacimab);Antibodies against tissue factor include Concizumab and Marstacimab;

TNF, 특히 TNFα 에 대한 항체로 인플릭시맙(Infliximab), 아달리무맙(Adalimumab), 골리무맙(Golimumab)과 항체 단편인 세르톨리주맙 페골(Certolizumab pegol), TNF와 알부민에 대한 이중항체인 오조랄리주맙(Ozoralizumab);Infliximab, Adalimumab, and Golimumab are antibodies against TNF, especially TNFα; Certolizumab pegol, an antibody fragment; and Ozo, a double antibody against TNF and albumin. Ozoralizumab;

VEGF에 대한 항체로 브롤루시주맙(Brolucizumab), 라니비주맙(Ranibizumab), 베바시주맙(Bevacizumab)과 VEGF와 Ang2의 이중 항체인 파리시맙(Faricimab);Antibodies against VEGF include Brolucizumab, Ranibizumab, and Bevacizumab, and Faricimab, a double antibody against VEGF and Ang2;

VEGF 수용체에 대한 항체로 라무시루맙(Ramucirumab); 및Ramucirumab, an antibody against the VEGF receptor; and

vWF에 대한 항체로 카플라시주맙(Caplacizumab).Caplacizumab, an antibody against vWF.

상기 '다클론 항체 의약품'은 면역글로불린(immune globulin) 등의 혈장 등에서 추출한 혈장 항체(serum antibody) 등이 바람직하나, 이에 한정되는 것은 아니다. The 'polyclonal antibody drug' is preferably a plasma antibody extracted from plasma such as immune globulin, but is not limited thereto.

본 발명에 따른 약학적 조성물에서 항체 의약품의 함량은 5 내지 500 mg/mL, 바람직하게는 20 내지 200 mg/mL, 더욱 바람직하게는 100 내지 150 mg/mL, 가장 바람직하게는 120±18 mg/mL 일 수 있으며, 예를 들어 약 110 mg/mL, 약 120 mg/mL 또는 약 130 mg/mL일 수 있다.The content of the antibody drug in the pharmaceutical composition according to the present invention is 5 to 500 mg/mL, preferably 20 to 200 mg/mL, more preferably 100 to 150 mg/mL, and most preferably 120 ± 18 mg/mL. mL, for example about 110 mg/mL, about 120 mg/mL or about 130 mg/mL.

본 발명에 따른 약학적 조성물에 있어서, 상기 '소분자 화합물'은 예방이나 치료의 목적으로 빠른 효과가 요구되는 약물이라면 제한 없이 사용 가능하다. 예를 들어, 모르핀(morphine) 계열의 진통제가 사용될 수 있다. 또한, 항암제에 의한 조직 괴사의 치료제로 사용하는 경우에 단독으로 사용하거나 antidote 약물인 Vinca Alkaloids 계통, Taxane 계통의 약물과 함께 사용할 수 있다. In the pharmaceutical composition according to the present invention, the 'small molecule compound' can be used without limitation as long as it is a drug that requires a rapid effect for the purpose of prevention or treatment. For example, a morphine-type painkiller may be used. In addition, when used as a treatment for tissue necrosis caused by anticancer drugs, it can be used alone or in combination with antidote drugs Vinca Alkaloids and Taxanes.

또한, 본 발명에 따른 약학적 조성물은 완충제, 안정화제, 및 계면활성제로 구성된 군에서 선택된 하나 이상을 추가로 포함할 수 있다. Additionally, the pharmaceutical composition according to the present invention may further include one or more selected from the group consisting of buffering agents, stabilizers, and surfactants.

본 발명에 따른 약학적 조성물에 있어서, 상기 '완충제'는 4 내지 8, 바람직하게는 5 내지 7의 pH를 제공하는 것이면 제한 없이 사용 가능하며, 바람직하게는 말산염(malate), 폼산염(formate), 시트르산염(citrate), 아세테이트(acetate), 프로피오네이트(propionate), 피리딘(pyridine), 피페라진(piperazine), 카코딜산(cacodylate), 석신산(succinate), 2-(N-모르폴리노)에탄설폰산(2-(N-morpholino)ethanesulfonic acid, MES), 히스티딘(histidine), 트리스(Tris), 비스-트리스(bis-Tris), 인산염(phosphate), 에탄올아민(ethanolamine), 탄산염(carbonate), 2-에테인술폰산(piperazine-N,N'-bis(2-ethanesulfonic acid), PIPES), 이미다졸(imidazole), 비스-트리스 프로판(BIS-Tris propane), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (Hydroxyethyl piperazine Ethane Sulfonic acid), 피로인산염(pyrophosphate), 및 트리에탄올아민(triethanolamine)으로 구성된 군에서 선택된 1종 이상이 바람직하고, 히스티딘 완충제, 예를 들면 L-히스티딘/HCl인 것이 보다 바람직하나, 이에 한정되는 것은 아니다. 상기 완충제의 농도는 0.001 내지 200 mM, 바람직하게는 1 내지 50 mM, 더욱 바람직하게는 5 내지 40 mM, 가장 바람직하게는 10 내지 30 mM일 수 있다.In the pharmaceutical composition according to the present invention, the 'buffer' can be used without limitation as long as it provides a pH of 4 to 8, preferably 5 to 7, and is preferably malate or formate. ), citrate, acetate, propionate, pyridine, piperazine, cacodylate, succinate, 2-(N-morpholy) 2-(N-morpholino)ethanesulfonic acid (MES), histidine, Tris, bis-Tris, phosphate, ethanolamine, carbonate (carbonate), 2-ethanesulfonic acid (piperazine-N,N'-bis(2-ethanesulfonic acid), PIPES), imidazole, BIS-Tris propane, BES (N,N- A group consisting of bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (Hydroxyethyl piperazine Ethane Sulfonic acid), pyrophosphate, and triethanolamine. One or more types selected from are preferred, and a histidine buffer, for example, L-histidine/HCl, is more preferred, but is not limited thereto. The concentration of the buffer may be 0.001 to 200mM, preferably 1 to 50mM, more preferably 5 to 40mM, and most preferably 10 to 30mM.

본 발명에 따른 약학적 조성물에 있어서, 상기 '안정화제'는 단백질의 안정화 목적으로 당업계에서 통상적으로 사용되는 물질이라면 제한 없이 사용 가능하며, 바람직한 예로서 탄수화물, 당류 또는 이들의 수화물, 당 알코올류 또는 이들의 수화물, 및 아미노산으로 구성된 군에서 선택된 1종 이상일 수 있다. In the pharmaceutical composition according to the present invention, the 'stabilizer' can be used without limitation as long as it is a substance commonly used in the art for the purpose of stabilizing proteins, and preferred examples include carbohydrates, sugars or their hydrates, and sugar alcohols. Or, it may be one or more selected from the group consisting of hydrates thereof, and amino acids.

상기 안정화제로 사용되는 '탄수화물, 당류 또는 당알코올류'는 트레할로스 또는 그의 수화물, 수크로스, 사카린, 글리세롤, 에리스톨, 트레이톨, 자일리톨, 아라비톨, 리비톨, 만니톨, 소르비톨, 갈락티톨, 푸시톨, 아이디톨, 이노시톨, 볼레미톨, 이소말트, 말티톨, 폴리글리시톨, 사이클로덱스트린(cyclodextrin), 하이드로옥실프로필 사이클로덱스트린(Hydroxypropyl Beta-cyclodextrin) 및 글루코스로 구성된 군에서 선택된 1종 이상일 수 있으나, 이에 한정되는 것은 아니며, 본 발명에 따른 약학적 조성물에서 안정화제로 사용되는 상기 당류 또는 당알코올류의 농도는 0.001 내지 500 mM, 바람직하게는 100 내지 300 mM, 더욱 바람직하게는 150 내지 250 mM, 가장 바람직하게는 180 내지 230 mM일 수 있다.'Carbohydrates, sugars or sugar alcohols' used as the stabilizer include trehalose or its hydrate, sucrose, saccharin, glycerol, erythol, threitol, xylitol, arabitol, ribitol, mannitol, sorbitol, galactitol, and fusitol. , iditol, inositol, volemitol, isomalt, maltitol, polyglycitol, cyclodextrin, hydroxypropyl cyclodextrin (Hydroxypropyl Beta-cyclodextrin), and glucose. It is not limited thereto, and the concentration of the sugars or sugar alcohols used as a stabilizer in the pharmaceutical composition according to the present invention is 0.001 to 500mM, preferably 100 to 300mM, more preferably 150 to 250mM, most preferably 150 to 250mM. Preferably it may be 180 to 230 mM.

상기 '아미노산'은 글루타민, 글루탐산, 글라이신, 라이신, 라이실라이신, 류신 메티오닌, 발린, 세린, 셀레노메티오닌, 시트롤린, 아르기닌, 아스파라진, 아스파트산, 오르니틴, 아이소류신, 타우린, 테아닌, 트레오닌, 트립토판, 타이로신, 페닐알라닌, 프롤린, 피롤라이신, 히스티딘 및 알라닌으로 구성된 군에서 선택된 1종 이상일 수 있으나, 이에 한정되는 것은 아니며, 본 발명에 따른 약학적 조성물에서 안정화제로 사용되는 아미노산의 농도는 1 내지 100 mM, 바람직하게는 3 내지 30 mM, 더욱 바람직하게는 5 내지 25 mM, 가장 바람직하게는 7 내지 20 mM일 수 있다.The 'amino acids' include glutamine, glutamic acid, glycine, lysine, lysyllysine, leucine, methionine, valine, serine, selenomethionine, citroline, arginine, asparagine, aspartic acid, ornithine, isoleucine, taurine, theanine, It may be one or more selected from the group consisting of threonine, tryptophan, tyrosine, phenylalanine, proline, pyrrolysine, histidine and alanine, but is not limited thereto, and the concentration of the amino acid used as a stabilizer in the pharmaceutical composition according to the present invention is It may be 1 to 100mM, preferably 3 to 30mM, more preferably 5 to 25mM, and most preferably 7 to 20mM.

본 발명에 따른 약학적 조성물에는 추가적으로 계면활성제가 포함될 수 있다. 상기 계면활성제는 폴리옥시에틸렌-소르비탄 지방산 에스테르 [폴리소르베이트(polysorbate) 또는 트윈(tween)], 폴리에틸렌-폴리프로필렌 글리콜, 폴리옥시에틸렌-스테아레이트, 폴리옥시에틸렌 알킬 에테르, 예를 들어, 폴리옥시에틸렌 모노라우릴 에테르, 알킬페닐폴리옥시에틸렌 에테르 [트리톤(triton)-X], 폴리옥시에틸렌-폴리옥시프로필렌 공중합체 [폴록사머(poloxamer), 플루로닉(pluronic)] 및 나트륨 도데실 설페이트(SDS) 등의 비이온성 계면활성제가 바람직하며, 더욱 바람직하게는 폴리소르베이트(polysorbate)가 사용될 수 있다. 상기 폴리소르베이트는 폴리소르베이트 20 또는 폴리소르베이트 80일 수 있다. 그러나, 그에 한정되는 것은 아니다. 본 발명에 따른 약학적 조성물에서 비이온성 계면활성제의 농도는 0.0000001% 내지 0.5%(w/v), 바람직하게는 0.000001% 내지 0.4%(w/v), 더욱 바람직하게는 0.00001% 내지 0.3%(w/v), 가장 바람직하게는 0.001% 내지 0.2% (w/v)일 수 있다.The pharmaceutical composition according to the present invention may additionally contain a surfactant. The surfactant is polyoxyethylene-sorbitan fatty acid ester (polysorbate or tween), polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ether, for example, poly Oxyethylene monolauryl ether, alkylphenylpolyoxyethylene ether [triton-X], polyoxyethylene-polyoxypropylene copolymer [poloxamer, pluronic] and sodium dodecyl sulfate. Nonionic surfactants such as (SDS) are preferred, and more preferably polysorbate can be used. The polysorbate may be polysorbate 20 or polysorbate 80. However, it is not limited thereto. The concentration of nonionic surfactant in the pharmaceutical composition according to the present invention is 0.0000001% to 0.5% (w/v), preferably 0.000001% to 0.4% (w/v), more preferably 0.00001% to 0.3% ( w/v), most preferably 0.001% to 0.2% (w/v).

본 발명에 따른 약학적 조성물은 정맥내 투여, 피하 투여, 근육 투여, 복강 투여, 내피 투여, 국소 투여, 비강내 투여, 폐내 투여 및 직장내 투여 등의 방법으로 투여할 수 있으며, 피하 투여를 통해 투여되는 것이 바람직하고, 피하 주사 투여를 위한 주사 제형의 형태로 사용되는 것이 더욱 바람직하다. 이에 따라 본 발명의 일 양태에서는, 본 발명에 따른 약학적 조성물을 포함하는 피하 투여용 제형을 제공한다.The pharmaceutical composition according to the present invention can be administered by methods such as intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration, and through subcutaneous administration. It is preferred to be administered, and it is more preferred to be used in the form of an injectable formulation for subcutaneous injection administration. Accordingly, in one aspect of the present invention, a formulation for subcutaneous administration containing the pharmaceutical composition according to the present invention is provided.

상기 피하 투여용 제형은 추가적인 희석 과정 없이 즉시 투여 가능한 형태(ready-to-injection) 형태로 제공될 수 있으며, 프리필드 시린지(pre-filled syringe) 또는 유리 앰플이나 플라스틱 용기에 포함되어 제공될 수 있다.The formulation for subcutaneous administration may be provided in a ready-to-injection form without additional dilution, and may be provided in a pre-filled syringe, glass ampoule, or plastic container. .

본 발명에 따른 약학적 조성물의 예방 또는 치료 대상이 되는 질환에는 특별한 제한은 없으며, 본 발명에 따른 PH-20 변이체와 병용 가능한 약물로 치료 가능한 질환이면 아무런 제한이 없다. 구체적으로 상기 질환은 암, 자가면역질환 등이 예시될 수 있으나, 이에 한정되는 것은 아니다.There are no particular restrictions on diseases that can be prevented or treated by the pharmaceutical composition according to the present invention, and there are no restrictions as long as it is a disease that can be treated with a drug that can be used in combination with the PH-20 variant according to the present invention. Specifically, the disease may include cancer, autoimmune disease, etc., but is not limited thereto.

상기 '암' 또는 '암종'은 특별히 제한되지 않으며, 고형암 및 혈액암을 모두 포함한다. 상기 암의 예로는 흑색종 등의 피부암, 간암, 간세포암(hepatocellular carcinoma), 위암, 유방암, 폐암, 난소암, 기관지암, 비인두암, 후두암, 췌장암, 방광암, 대장암, 결장암, 자궁경부암, 뇌암, 전립선암, 골암, 갑상선암, 부갑상선암, 신장암, 식도암, 담도암, 고환암, 직장암, 두경부암, 경추암, 요관암, 골육종, 신경아세포종, 섬유육종, 횡문근육종, 성상세포종, 신경모세포종 및 신경교종으로 이루어진 군에서 선택될 수 있고, 바람직하게는 위암, 대장암, 유방암, 폐암 및 신장암으로 구성된 군에서 선택될 수 있지만, 이에 한정되는 것은 아니다.The term ‘cancer’ or ‘carcinoma’ is not particularly limited and includes both solid cancer and blood cancer. Examples of the above cancer include skin cancer such as melanoma, liver cancer, hepatocellular carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, cervical cancer, and brain cancer. , prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma and nerve. It may be selected from the group consisting of glioma, and preferably may be selected from the group consisting of stomach cancer, colon cancer, breast cancer, lung cancer, and kidney cancer, but is not limited thereto.

상기 '자가면역질환'은 류마티스 관절염(rheumatoid arthritis), 천식(asthma), 건선(psoriasis), 다발성 경화증(multiple sclerosis), 알레르기성 비염(allergic rhinitis), 크론병(Crohn's disease), 궤양성 대장염(ulcerative colitis), 전신성 홍반성 루푸스, 제1형 당뇨병(type I diabetes), 염증성 장질환(Inflammatory bowel disease, IBD) 및 아토피성 피부염(Atopic dermatitis)으로 구성된 군에서 선택될 수 있지만 이에 한정되는 것은 아니다.The 'autoimmune disease' includes rheumatoid arthritis, asthma, psoriasis, multiple sclerosis, allergic rhinitis, Crohn's disease, and ulcerative colitis ( ulcerative colitis, systemic lupus erythematosus, type I diabetes, inflammatory bowel disease (IBD), and atopic dermatitis. .

따라서, 본 발명은 서열번호 1, 서열번호 2, 및 서열번호 3으로 이루어진 군에서 선택된 어느 하나의 신규한 히알루로니다제 PH-20 변이체, 상기 변이체를 암호화하는 폴리뉴클레오티드, 상기 폴리펩티드를 포함하는 발현 벡터, 상기 발현 벡터를 포함하는 숙주 세포주, 상기 숙주 세포주를 배양하는 단계를 포함하는 히알루로니다제 PH-20 변이체의 제조 방법, 및 상기 히알루로니다제 PH-20 변이체를 포함하는 질환의 예방 또는 치료용 약학적 조성물을 제공한다. 본 발명에 따른 신규한 히알루로니다제 PH-20 변이체는 야생형의 인간 히알루로니다제 PH-20와 비교하여 안정성이 높고 활성이 뛰어나므로, 이와 함께 사용되는 약물의 치료 효과를 극대화할 수 있다. Accordingly, the present invention provides a novel hyaluronidase PH-20 variant selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, a polynucleotide encoding the variant, and an expression comprising the polypeptide. A vector, a host cell line containing the expression vector, a method for producing a hyaluronidase PH-20 variant comprising culturing the host cell line, and prevention of diseases containing the hyaluronidase PH-20 variant, or A pharmaceutical composition for therapeutic use is provided. The novel hyaluronidase PH-20 variant according to the present invention has high stability and excellent activity compared to wild-type human hyaluronidase PH-20, and thus can maximize the therapeutic effect of drugs used together with it.

도 1은 본 발명의 히알루로니다제 PH-20 변이체 Hy1에 있어서, Y57H, W304H, T306R, L307K, 및 T306A 아미노산 잔기의 치환 위치를 도식화한 것이다.
도 2는 본 발명의 히알루로니다제 PH-20 변이체 Hy1에 있어서 이황화결합의 위치를 도식화한 것이다.
도 3은 바이오시스템 社의 Protein Thermal Shift™ Dye Kit를 사용하여 히알루로니다제 PH-20 변이체 Hy1, Hy1-M1, 및 Hy2의 TM 데이터를 나타낸 것이다. 전체 변이체에 대한 자료는 표 4에 기재되었다.
도 4는 히알루로니다제 PH-20 변이체들 중 일부에 대하여 37℃에서 효소 안정성을 평가한 결과를 나타낸 것이다. 전체 변이체에 대한 자료는 표 5에 기재되어 있다.
도 5는 히알루로니다제 PH-20 변이체 중 일부에 대하여 50℃에서 효소 안정성을 평가한 결과를 나타낸 것이다. 전체 변이체에 대한 자료는 표 6에 기재되어 있다.Figure 1 schematically illustrates the substitution positions of amino acid residues Y57H, W304H, T306R, L307K, and T306A in the hyaluronidase PH-20 variant Hy1 of the present invention.
Figure 2 schematically shows the position of the disulfide bond in the hyaluronidase PH-20 variant Hy1 of the present invention.
Figure 3 shows TM data of hyaluronidase PH-20 variants Hy1, Hy1-M1, and Hy2 using Biosystem's Protein Thermal Shift™ Dye Kit. Data for all variants are listed in Table 4.
Figure 4 shows the results of evaluating enzyme stability at 37°C for some of the hyaluronidase PH-20 variants. Data for all variants are listed in Table 5.
Figure 5 shows the results of evaluating enzyme stability at 50°C for some of the hyaluronidase PH-20 variants. Data for all variants are listed in Table 6.

이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

실시예 1. 히알루로니다제 PH-20 구조의 3D 모델링Example 1. 3D modeling of hyaluronidase PH-20 structure

1-1. Active site 모델링1-1. Active site modeling

단백질 구조가 밝혀지지 않은 히알루로니다제 PH-20 에 대해 DeepMind 社의 알파폴드2 모델링 프로그램을 이용하여 초기 모델 구조 5개를 제조하였다. 이후, 5개의 모델을 주형(template) 구조로 사용하고, ㈜굿인텔리전스의 단백질 구조 모델링 기법을 통해 최종 3차 구조를 완성하였다. For hyaluronidase PH-20, the protein structure of which has not been revealed, five initial model structures were prepared using DeepMind's AlphaFold2 modeling program. Afterwards, five models were used as template structures, and the final tertiary structure was completed through Good Intelligence Co., Ltd.'s protein structure modeling technique.

Active site의 아미노산 잔기들에 대해서는 19개의 다른 아미노산으로의 치환을 통해 여러 변이체를 만들고 각각의 변이체들에 대해 단백질과 리간드에 대한 결합력 예측을 위해 다양한 예측 프로그램을 사용하였다. 최종적으로 이 중 결합력이 높을 것으로 예측되는 변이체 5개를 선별하여 제작하였다.For the amino acid residues in the active site, several variants were created by substituting 19 different amino acids, and various prediction programs were used to predict the binding affinity for proteins and ligands for each variant. Finally, five variants predicted to have high binding affinity were selected and produced.

1-2. 이황화결합 모델링1-2. Disulfide bond modeling

Active site 모델링을 진행한 후 효소의 안정도를 증가시키기 위하여 내부 이황화결합을 도입하는 작업을 수행하였다. 이황화 결합 예측(disulfide predict)은 YASARA Web server를 이용하였다. 이후 제안된 목록들 중에서 유효한 변이체를 선정하기 위하여 이황화결합이 가능한 거리를 측정해주는 Protein contact map visualization (Andreas Viklund.)을 이용하여 이황화결합 모델링을 수행하였다.After active site modeling was performed, an internal disulfide bond was introduced to increase the stability of the enzyme. Disulfide bond prediction was performed using the YASARA Web server. Afterwards, in order to select valid variants from the proposed list, disulfide bond modeling was performed using Protein contact map visualization (Andreas Viklund.), which measures the distance between possible disulfide bonds.

실시예 2. 히알루로니다제 PH-20 변이체의 제작Example 2. Construction of hyaluronidase PH-20 variant

상기 모델링 및 시뮬레이션 결과를 바탕으로 히알루로니다제 PH-20 변이체를 제작하였다. Based on the above modeling and simulation results, a hyaluronidase PH-20 variant was produced.

2-1. Cloning vector 제작2-1. Cloning vector production

일반적인 mammalian cell 단백질 발현 벡터와 일반적인 발현용 mammalian cell 균주를 사용하였으며, Cloning용 E. coli 균주는 Top10을 사용하였다. ㈜코스모진텍에서 유전자 합성, primer 주문 제작을 진행하였다. 유전자 재조합 시 사용된 제한효소는 모두 enzynomics의 제품이며, ligase는 enzynomics의 EZ-Fusion HT Cloning Kit이다. Insert PCR, point mutation에 사용된 DNA polymerase는 Takara의 PrimeSTAR HS이다. 사용된 DNA gel extraction kit, plasmid mini prep kit는 ㈜코스모진텍의 제품이다. DNA sequencing은 ㈜마크로젠에 의뢰하여 수행하였다.A general mammalian cell protein expression vector and a general mammalian cell strain for expression were used, and Top10 E. coli strains for cloning were used. Cosmogenetech Co., Ltd. conducted gene synthesis and custom-made primers. The restriction enzymes used during genetic recombination are all from Enzynomics, and the ligase is Enzynomics' EZ-Fusion HT Cloning Kit. The DNA polymerase used for insert PCR and point mutation is Takara's PrimeSTAR HS. The DNA gel extraction kit and plasmid mini prep kit used are products of Cosmogenetech Co., Ltd. DNA sequencing was performed by requesting Macrogen Co., Ltd.

사용된 primer는 표 2과 같다.The primers used are shown in Table 2.

Mutation
NameMutation
Name primer (F)primer (F) primer (R)primer (R) Hy1, Hy2, Hy3Hy1, Hy2, Hy3 taccgagctcggatccgccaccattaccgagctcggatccgccaccat gggccctctagactcgagtcagtgatggggccctctagactcgagtcagtgatg Y57HY57H ggtgttacaatatttcacgttgatagacttggctacggtgttacaatatttcacgttgatagacttggctac gtagccaagtctatcaacgtgaaatattgtaacaccgtagccaagtctatcaacgtgaaatattgtaacacc W304HW304H tctggaattgtaatacacggaaccctcagtataatgtctggaattgtaatacacggaaccctcagtataatg cattatactgagggttccgtgtattacaattccagacattatactgagggttccgtgtattacaattccaga T306RT306R ggaattgtaatatggggaagactcagtataatgcgaggaattgtaatatggggaagactcagtataatgcga tcgcattatactgagtcttccccatattacaattcctcgcattatactgagtcttccccatattacaattcc L307KL307K ggaattgtaatatggggaaccaagagtataatgcgaaggaattgtaatatggggaaccaagagtataatgcgaa ttcgcattatactcttggttccccatattacaattccttcgcattatactcttggttccccatattacaattcc T306AT306A attgtaatatggggagccctcagtataatgcgaattgtaatatggggagccctcagtataatgcga tcgcattatactgagggctccccatattacaattcgcattatactgagggctccccatattacaat P80CP80C gtgaatggaggaatctgccagaagatttccttagtgaatggaggaatctgccagaagatttcctta aaggaaatcttctggcagattcctccattcacaaggaaatcttctctggcagattcctccattcac A160CA160C caagaatttgaaaagtgcgggaaggatttcctgcaagaatttgaaaagtgcgggaaggatttcctg caggaaatccttcccgcacttttcaaattcttgcaggaaatccttcccgcacttttcaaattcttg G291CG291C cttgtgtatacattttgcgaaactgttgctctggcttgtgtatacattttgcgaaactgttgctctgg ccagagcaacagtttcgcaaaatgtatacacaagccagagcaacagtttcgcaaaatgtatacacaag Y362CY362C tggaattcaagtgactgccttcacctcaatggaattcaagtgactgccttcacctcaa gaaggcagtcacttgaattccagtttttccgaaggcagtcacttgaattccagtttttcc L180CL180C cttcggccaaatcactgctggggttattatcttcggccaaatcactgctggggttattat ataataaccccagcagtgatttggccgaagataataaccccagcagtgatttggccgaag A223CA223C tggaatgaaagcacttgcctttacccatccatttggaatgaaagcacttgcctttacccatccatt ggatgggtaaaggcaagtgctttcattccggatgggtaaaggcaagtgctttcattcc P6CP6C ctgaatttcagagcatgccctgttattccaaatgtctgaatttcagagcatgccctgttattccaaatgt acatttggaataacagggcatgctctgaaattcagacatttggaataacagggcatgctctgaaattcag Y403CY403C ttttattgcagctgttgcagcaccttgagttgtttttatgcagctgttgcagcaccttgagttgt acaactcaaggtgctgcaacagctgcaataaaaacaactcaaggtgctgcaacagctgcaataaaa S36CS36C gagccactagatatgtgcctcttctctttcatagagccactagatatgtgcctcttctctttcata atgaaagagaagaggcacatatctagtggctcatgaaagagaagaggcacatatctagtggctc D426CD426C gatgtgtgtattgcttgcggtgtctgtatagatgatgtgtgtattgcttgcggtgtctgtatagat atctatacagacaccgcaagcaatacacacatcatctatacagacaccgcaagcaatacacacatc

2-2. 단백질 발현2-2. protein expression

ExpiFectamine CHO reagent를 이용하여 Cricetulus griseus(CHO)에 유전자를 형질전환 시킨 후 습도 80%, 5~8% CO2 의 37℃ 궤도형 쉐이커에서 33~38 ml 세포를 9~13일간 배양하였다. 발현된 단백질은 SDS-page와 western blot을 이용하여 확인 한 후 0.22㎛ 필터로 여과하여 정제에 사용하였다.After gene transformation into Cricetulus griseus (CHO) using ExpiFectamine CHO reagent, 33-38 ml cells were cultured for 9-13 days in an orbital shaker at 37°C with 80% humidity and 5-8% CO2. The expressed protein was confirmed using SDS-page and western blot, then filtered through a 0.22㎛ filter and used for purification.

2-3. 단백질 정제2-3. protein purification

정제는 HisTrap HP 컬럼을 이용하였다. 컬럼은 사용 전 binding buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄)를 이용하여 평형을 맞춰주었다. 준비된 컬럼에 준비한 배양액을 흘려주어 목적 단백질을 컬럼에 결합시킨 뒤 binding buffer로 다시 한번 세척해주었다. 이후 컬럼에 결합 된 목적 단백질을 용출하기 위해 elution buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.5 M imidazole를 이용, linear gradient 방법으로 목적 단백질을 선별하였다. 모든 과정은 3 ml/min 속도로 진행되었다. 선별한 목적 단백질은 0.2 ㎛ 필터로 여과 후 활성을 측정하였다. FPLC는 GE UPC-800을 사용하였다.Purification was performed using a HisTrap HP column. The column was equilibrated using binding buffer (137mM NaCl, 2.7mM KCl, 10mM Na ₂ HPO ₄ , 1.8mM KH ₂ PO ₄ ) before use. The prepared culture medium was flowed through the prepared column to bind the target protein to the column, and then washed again with binding buffer. Afterwards, to elute the target protein bound to the column, elution buffer (137mM NaCl, 2.7mM KCl, _{10mM Na2HPO4} , _{1.8mMKH2PO4 ,} 0.5M imidazole) was used to select the target protein using the linear gradient method. All processes were carried out at a rate of 3 ml/min. The selected target protein was filtered through a 0.2 ㎛ filter and its activity was measured. FPLC was performed using a GE UPC-800.

실시예 3. 히알루로니다제 PH-20 변이체의 활성 평가Example 3. Activity evaluation of hyaluronidase PH-20 variants

히알루로니다제 효소 활성을 측정하는 Turbidimetric assay 방법은 반응 용액에 잔존하는 히알루론산이 산성화된 알부민(BSA)과 결합하여 응집체를 형성하는 정도를 흡광도로 측정하는 방법으로, 히알루로니다제에 의해 히알루론산이 가수분해되면 알부민과 결합하는 양이 감소하여 흡광도가 감소하는 원리를 이용하여 활성을 측정하였다. 야생형 히알루로니다제를 cold enzyme diluent buffer를 이용하여 적합한 농도(unit/mL)가 되도록 희석하여 각 튜브에 준비하였다. 정제된 히알루로니다제 시료를 cold enzyme diluent buffer를 이용, 적합한 농도로 희석하여 각 튜브에 준비하였다. 새로운 튜브에 3 mg/mL인 히알루론산 용액을 농도가 0.3 mg/mL 되게 10배 희석하여 각 튜브의 부피가 180 ㎕가 되게 하였다. 희석한 히알루론산 용액에 히알루로니다제가 포함된 시료를 60 ㎕ 넣고 혼합(hyaluronic acid : hyaluronidase=3:1)하여 37℃에서 45분간 반응시켰다. 반응이 끝나면 96-well plate에 반응시킨 히알루로니다제 50 ㎕와 acidic albumin solution 250 ㎕를 각 well에 넣고 실온에서 10분간 진탕한 후 600 nm에서 분광 광도계로 흡광도를 측정하였다. 야생형 히알루로니다제로부터 얻은 흡광도를 이용하여 standard curve를 만들었다. Standard curve를 이용하여 히알루로니다제 시료의 활성(units/mL)을 구하였다. 각 히알루로니다제 활성은 3회 반복으로부터 얻은 값과 오차값을 표기하였다.Turbidimetric assay to measure hyaluronidase enzyme activity is a method of measuring the degree to which hyaluronic acid remaining in the reaction solution combines with acidified albumin (BSA) to form aggregates using absorbance. Hyaluronic acid is absorbed by hyaluronidase. Activity was measured using the principle that when ronic acid is hydrolyzed, the amount bound to albumin decreases and the absorbance decreases. Wild-type hyaluronidase was diluted to an appropriate concentration (unit/mL) using cold enzyme diluent buffer and prepared in each tube. The purified hyaluronidase sample was diluted to an appropriate concentration using cold enzyme diluent buffer and prepared in each tube. In a new tube, the 3 mg/mL hyaluronic acid solution was diluted 10 times to a concentration of 0.3 mg/mL, so that the volume of each tube was 180 ㎕. 60 ㎕ of a sample containing hyaluronidase was added to the diluted hyaluronic acid solution, mixed (hyaluronic acid: hyaluronidase=3:1), and reacted at 37°C for 45 minutes. After the reaction was completed, 50 μl of the reacted hyaluronidase and 250 μl of the acidic albumin solution were added to each well in a 96-well plate, shaken for 10 minutes at room temperature, and the absorbance was measured with a spectrophotometer at 600 nm. A standard curve was created using the absorbance obtained from wild-type hyaluronidase. The activity (units/mL) of the hyaluronidase sample was calculated using the standard curve. For each hyaluronidase activity, the value and error value obtained from three repetitions were indicated.

그 결과, 각 히알루로니다제 PH-20 변이체의 활성은 하기 표 3과 같았다.As a result, the activity of each hyaluronidase PH-20 variant was shown in Table 3 below.

서열번호sequence number 명칭designation 돌연변이mutation Exp. ActExp. Act 22 Hy1Hy1 　 1.000 1.000 33 Hy2Hy2 　 2.105 2.105 44 Hy3Hy3 　 3.442 3.442 55 Hy1-M1Hy1-M1 P80C, A160CP80C, A160C 2.780 2.780 66 Hy1-M2Hy1-M2 G291C, Y362CG291C, Y362C 1.028 1.028 77 Hy1-M3Hy1-M3 L180C, A223CL180C, A223C 2.663 2.663 88 Hy1-M4Hy1-M4 P6C, Y403CP6C, Y403C 1.001 1.001 99 Hy1-M5Hy1-M5 S36C, D426CS36C, D426C 1.637 1.637 1010 Hy1-A1Hy1-A1 Y57HY57H 1.049 1.049 1111 Hy1-A1-M1Hy1-A1-M1 Y57H, P80C, A160CY57H, P80C, A160C 2.916 2.916 1212 Hy1-A1-M2Hy1-A1-M2 Y57H, G291C, Y362CY57H, G291C, Y362C 1.078 1.078 1313 Hy1-A1-M3Hy1-A1-M3 Y57H, L180C, A223CY57H, L180C, A223C 2.793 2.793 1414 Hy1-A1-M4Hy1-A1-M4 Y57H, P6C, Y403CY57H, P6C, Y403C 1.050 1.050 1515 Hy1-A1-M5Hy1-A1-M5 Y57H, S36C, D426CY57H, S36C, D426C 1.717 1.717 1616 Hy1-A2Hy1-A2 W304HW304H 1.013 1.013 1717 Hy1-A2-M1Hy1-A2-M1 W304H, P80C, A160CW304H, P80C, A160C 2.816 2.816 1818 Hy1-A2-M2Hy1-A2-M2 W304H, G291C, Y362CW304H, G291C, Y362C 1.042 1.042 1919 Hy1-A2-M3Hy1-A2-M3 W304H, L180C, A223CW304H, L180C, A223C 2.698 2.698 2020 Hy1-A2-M4Hy1-A2-M4 W304H, P6C, Y403CW304H, P6C, Y403C 1.014 1.014 2121 Hy1-A2-M5Hy1-A2-M5 W304H, S36C, D426CW304H, S36C, D426C 1.658 1.658 2222 Hy1-A3Hy1-A3 T306RT306R 1.464 1.464 2323 Hy1-A3-M1Hy1-A3-M1 T306R, P80C, A160CT306R, P80C, A160C 4.070 4.070 2424 Hy1-A3-M2Hy1-A3-M2 T306R, G291C, Y362CT306R, G291C, Y362C 1.505 1.505 2525 Hy1-A3-M3Hy1-A3-M3 T306R, L180C, A223CT306R, L180C, A223C 3.898 3.898 2626 Hy1-A3-M4Hy1-A3-M4 T306R, P6C, Y403CT306R, P6C, Y403C 1.465 1.465 2727 Hy1-A3-M5Hy1-A3-M5 T306R, S36C, D426CT306R, S36C, D426C 2.396 2.396 2828 Hy1-A4Hy1-A4 L307KL307K 0.405 0.405 2929 Hy1-A4-M1Hy1-A4-M1 L307K, P80C, A160CL307K, P80C, A160C 1.125 1.125 3030 Hy1-A4-M2Hy1-A4-M2 L307K, G291C, Y362CL307K, G291C, Y362C 0.416 0.416 3131 Hy1-A4-M3Hy1-A4-M3 L307K, L180C, A223CL307K, L180C, A223C 1.077 1.077 3232 Hy1-A4-M4Hy1-A4-M4 L307K, P6C, Y403CL307K, P6C, Y403C 0.405 0.405 3333 Hy1-A4-M5Hy1-A4-M5 L307K, S36C, D426CL307K, S36C, D426C 0.662 0.662 3434 Hy1-A5Hy1-A5 T306AT306A 1.144 1.144 3535 Hy1-A5-M1Hy1-A5-M1 T306A, P80C, A160CT306A, P80C, A160C 3.180 3.180 3636 Hy1-A5-M2Hy1-A5-M2 T306A, G291C, Y362CT306A, G291C, Y362C 1.176 1.176 3737 Hy1-A5-M3Hy1-A5-M3 T306A, L180C, A223CT306A, L180C, A223C 3.045 3.045 3838 Hy1-A5-M4Hy1-A5-M4 T306A, P6C, Y403CT306A, P6C, Y403C 1.145 1.145 3939 Hy1-A5-M5Hy1-A5-M5 T306A, S36C, D426CT306A, S36C, D426C 1.872 1.872 4040 Hy2-M1Hy2-M1 P80C, A160CP80C, A160C 5.851 5.851 4141 Hy2-M2Hy2-M2 G291C, Y362CG291C, Y362C 2.164 2.164 4242 Hy2-M3Hy2-M3 L180C, A223CL180C, A223C 5.604 5.604 4343 Hy2-M4Hy2-M4 P6C, Y403CP6C,Y403C 2.107 2.107 4444 Hy2-M5Hy2-M5 S36C, D426CS36C, D426C 3.445 3.445 4545 Hy2-A1Hy2-A1 Y57HY57H 2.207 2.207 4646 Hy2-A1-M1Hy2-A1-M1 Y57H, P80C, A160CY57H, P80C, A160C 6.137 6.137 4747 Hy2-A1-M2Hy2-A1-M2 Y57H, G291C, Y362CY57H, G291C, Y362C 2.270 2.270 4848 Hy2-A1-M3Hy2-A1-M3 Y57H, L180C, A223CY57H, L180C, A223C 5.878 5.878 4949 Hy2-A1-M4Hy2-A1-M4 Y57H, P6C, Y403CY57H, P6C, Y403C 2.210 2.210 5050 Hy2-A1-M5Hy2-A1-M5 Y57H, S36C, D426CY57H, S36C, D426C 3.614 3.614 5151 Hy2-A2Hy2-A2 W304HW304H 2.132 2.132 5252 Hy2-A2-M1Hy2-A2-M1 W304H, P80C, A160CW304H, P80C, A160C 5.928 5.928 5353 Hy2-A2-M2Hy2-A2-M2 W304H, G291C, Y362CW304H, G291C, Y362C 2.192 2.192 5454 Hy2-A2-M3Hy2-A2-M3 W304H, L180C, A223CW304H, L180C, A223C 5.677 5.677 5555 Hy2-A2-M4Hy2-A2-M4 W304H, P6C, Y403CW304H, P6C, Y403C 2.134 2.134 5656 Hy2-A2-M5Hy2-A2-M5 W304H, S36C, D426CW304H, S36C, D426C 3.490 3.490 5757 Hy2-A3Hy2-A3 T306RT306R 3.081 3.081 5858 Hy2-A3-M1Hy2-A3-M1 T306R, P80C, A160CT306R, P80C, A160C 8.565 8.565 5959 Hy2-A3-M2Hy2-A3-M2 T306R, G291C, Y362CT306R, G291C, Y362C 3.167 3.167 6060 Hy2-A3-M3Hy2-A3-M3 T306R, L180C, A223CT306R, L180C, A223C 8.203 8.203 6161 Hy2-A3-M4Hy2-A3-M4 T306R, P6C, Y403CT306R, P6C, Y403C 3.084 3.084 6262 Hy2-A3-M5Hy2-A3-M5 T306R, S36C, D426CT306R, S36C, D426C 5.043 5.043 6363 Hy2-A4Hy2-A4 L307KL307K 0.852 0.852 6464 Hy2-A4-M1Hy2-A4-M1 L307K, P80C, A160CL307K, P80C, A160C 2.367 2.367 6565 Hy2-A4-M2Hy2-A4-M2 L307K, G291C, Y362CL307K, G291C, Y362C 0.876 0.876 6666 Hy2-A4-M3Hy2-A4-M3 L307K, L180C, A223CL307K, L180C, A223C 2.268 2.268 6767 Hy2-A4-M4Hy2-A4-M4 L307K, P6C, Y403CL307K, P6C, Y403C 0.852 0.852 6868 Hy2-A4-M5Hy2-A4-M5 L307K, S36C, D426CL307K, S36C, D426C 1.394 1.394 6969 Hy2-A5Hy2-A5 T306AT306A 2.407 2.407 7070 Hy2-A5-M1Hy2-A5-M1 T306A, P80C, A160CT306A, P80C, A160C 6.692 6.692 7171 Hy2-A5-M2Hy2-A5-M2 T306A, G291C, Y362CT306A, G291C, Y362C 2.475 2.475 7272 Hy2-A5-M3Hy2-A5-M3 T306A, L180C, A223CT306A, L180C, A223C 6.409 6.409 7373 Hy2-A5-M4Hy2-A5-M4 T306A, P6C, Y403CT306A, P6C, Y403C 2.409 2.409 7474 Hy2-A5-M5Hy2-A5-M5 T306A, S36C, D426CT306A, S36C, D426C 3.940 3.940 7575 Hy3-M1Hy3-M1 P80C, A160CP80C, A160C 9.568 9.568 7676 Hy3-M2Hy3-M2 G291C, Y362CG291C, Y362C 3.539 3.539 7777 Hy3-M3Hy3-M3 L180C, A223CL180C, A223C 9.165 9.165 7878 Hy3-M4Hy3-M4 P6C, Y403CP6C, Y403C 3.445 3.445 7979 Hy3-M5Hy3-M5 S36C, D426CS36C, D426C 5.634 5.634 8080 Hy3-A1Hy3-A1 Y57HY57H 3.610 3.610 8181 Hy3-A1-M1Hy3-A1-M1 Y57H, P80C, A160CY57H, P80C, A160C 10.036 10.036 8282 Hy3-A1-M2Hy3-A1-M2 Y57H, G291C, Y362CY57H, G291C, Y362C 3.712 3.712 8383 Hy3-A1-M3Hy3-A1-M3 Y57H, L180C, A223CY57H, L180C, A223C 9.613 9.613 8484 Hy3-A1-M4Hy3-A1-M4 Y57H, P6C, Y403CY57H, P6C, Y403C 3.613 3.613 8585 Hy3-A1-M5Hy3-A1-M5 Y57H, S36C, D426CY57H, S36C, D426C 5.909 5.909 8686 Hy3-A2Hy3-A2 W304HW304H 3.486 3.486 8787 Hy3-A2-M1Hy3-A2-M1 W304H, P80C, A160CW304H, P80C, A160C 9.693 9.693 8888 Hy3-A2-M2Hy3-A2-M2 W304H, G291C, Y362CW304H, G291C, Y362C 3.585 3.585 8989 Hy3-A2-M3Hy3-A2-M3 W304H, L180C, A223CW304H, L180C, A223C 9.284 9.284 9090 Hy3-A2-M4Hy3-A2-M4 W304H, P6C, Y403CW304H, P6C, Y403C 3.490 3.490 9191 Hy3-A2-M5Hy3-A2-M5 W304H, S36C, D426CW304H, S36C, D426C 5.707 5.707 9292 Hy3-A3Hy3-A3 T306RT306R 5.038 5.038 9393 Hy3-A3-M1Hy3-A3-M1 T306R, P80C, A160CT306R, P80C, A160C 14.006 14.006 9494 Hy3-A3-M2Hy3-A3-M2 T306R, G291C, Y362CT306R, G291C, Y362C 5.180 5.180 9595 Hy3-A3-M3Hy3-A3-M3 T306R, L180C, A223CT306R, L180C, A223C 13.415 13.415 9696 Hy3-A3-M4Hy3-A3-M4 T306R, P6C, Y403CT306R, P6C, Y403C 5.043 5.043 9797 Hy3-A3-M5Hy3-A3-M5 T306R, S36C, D426CT306R, S36C, D426C 8.247 8.247 9898 Hy3-A4Hy3-A4 L307KL307K 1.392 1.392 9999 Hy3-A4-M1Hy3-A4-M1 L307K, P80C, A160CL307K, P80C, A160C 3.871 3.871 100100 Hy3-A4-M2Hy3-A4-M2 L307K, G291C, Y362CL307K, G291C, Y362C 1.432 1.432 101101 Hy3-A4-M3Hy3-A4-M3 L307K, L180C, A223CL307K, L180C, A223C 3.708 3.708 102102 Hy3-A4-M4Hy3-A4-M4 L307K, P6C, Y403CL307K, P6C, Y403C 1.394 1.394 103103 Hy3-A4-M5Hy3-A4-M5 L307K, S36C, D426CL307K, S36C, D426C 2.280 2.280 104104 Hy3-A5Hy3-A5 T306AT306A 3.936 3.936 105105 Hy3-A5-M1Hy3-A5-M1 T306A, P80C, A160CT306A, P80C, A160C 10.943 10.943 106106 Hy3-A5-M2Hy3-A5-M2 T306A, G291C, Y362CT306A, G291C, Y362C 4.047 4.047 107107 Hy3-A5-M3Hy3-A5-M3 T306A, L180C, A223CT306A, L180C, A223C 10.481 10.481 108108 Hy3-A5-M4Hy3-A5-M4 T306A, P6C, Y403CT306A, P6C, Y403C 3.940 3.940 109109 Hy3-A5-M5Hy3-A5-M5 T306A, S36C, D426CT306A, S36C, D426C 6.443 6.443

실시예 4. 히알루로니다제 PH-20 변이체의 Tm 분석Example 4. Tm analysis of hyaluronidase PH-20 variants

바이오시스템 社의 Protein Thermal Shift™ Dye Kit를 사용하여 히알루로니다제 PH-20 변이체의 Tm 데이터를 확보하였다. 구체적으로는 변이체들의 열안정성을 모니터링하기 위하여 노출된 소수성 잔류물에 결합하는 염료를 이용하였고 real-time PCR 기기로 측정하였다.Tm data of hyaluronidase PH-20 variant was obtained using Biosystem's Protein Thermal Shift™ Dye Kit. Specifically, to monitor the thermal stability of the variants, a dye that binds to exposed hydrophobic residues was used and measured using a real-time PCR device.

그 결과, 측정된 각 히알루로니다제 PH-20 변이체의 Tm 데이터는 하기의 표 4에 기재된 바와 같았으며, 그 중 Hy1, Hy1-M1 및 Hy2에 대하여 도 3에 도시하였다.As a result, the Tm data of each hyaluronidase PH-20 variant measured were as shown in Table 4 below, and among them, Hy1, Hy1-M1, and Hy2 are shown in Figure 3.

서열번호sequence number 명칭designation 돌연변이mutation TMTM 22 Hy1Hy1 62.0 62.0 33 Hy2Hy2 61.5 61.5 44 Hy3Hy3 62.6 62.6 55 Hy1-M1Hy1-M1 P80C, A160CP80C, A160C 62.4 62.4 66 Hy1-M2Hy1-M2 G291C, Y362CG291C, Y362C 61.5 61.5 77 Hy1-M3Hy1-M3 L180C, A223CL180C, A223C 62.4 62.4 88 Hy1-M4Hy1-M4 P6C, Y403CP6C, Y403C 61.5 61.5 99 Hy1-M5Hy1-M5 S36C, D426CS36C, D426C 61.9 61.9 1010 Hy1-A1Hy1-A1 Y57HY57H 62.5 62.5 1111 Hy1-A1-M1Hy1-A1-M1 Y57H, P80C, A160CY57H, P80C, A160C 62.4 62.4 1212 Hy1-A1-M2Hy1-A1-M2 Y57H, G291C, Y362CY57H, G291C, Y362C 61.5 61.5 1313 Hy1-A1-M3Hy1-A1-M3 Y57H, L180C, A223CY57H, L180C, A223C 62.4 62.4 1414 Hy1-A1-M4Hy1-A1-M4 Y57H, P6C, Y403CY57H, P6C, Y403C 61.5 61.5 1515 Hy1-A1-M5Hy1-A1-M5 Y57H, S36C, D426CY57H, S36C, D426C 62.0 62.0 1616 Hy1-A2Hy1-A2 W304HW304H 61.5 61.5 1717 Hy1-A2-M1Hy1-A2-M1 W304H, P80C, A160CW304H, P80C, A160C 62.4 62.4 1818 Hy1-A2-M2Hy1-A2-M2 W304H, G291C, Y362CW304H, G291C, Y362C 61.5 61.5 1919 Hy1-A2-M3Hy1-A2-M3 W304H, L180C, A223CW304H, L180C, A223C 62.4 62.4 2020 Hy1-A2-M4Hy1-A2-M4 W304H, P6C, Y403CW304H, P6C, Y403C 61.5 61.5 2121 Hy1-A2-M5Hy1-A2-M5 W304H, S36C, D426CW304H, S36C, D426C 61.9 61.9 2222 Hy1-A3Hy1-A3 T306RT306R 61.8 61.8 2323 Hy1-A3-M1Hy1-A3-M1 T306R, P80C, A160CT306R, P80C, A160C 62.7 62.7 2424 Hy1-A3-M2Hy1-A3-M2 T306R, G291C, Y362CT306R, G291C, Y362C 61.8 61.8 2525 Hy1-A3-M3Hy1-A3-M3 T306R, L180C, A223CT306R, L180C, A223C 62.7 62.7 2626 Hy1-A3-M4Hy1-A3-M4 T306R, P6C, Y403CT306R, P6C, Y403C 61.8 61.8 2727 Hy1-A3-M5Hy1-A3-M5 T306R, S36C, D426CT306R, S36C, D426C 62.3 62.3 2828 Hy1-A4Hy1-A4 L307KL307K 60.6 60.6 2929 Hy1-A4-M1Hy1-A4-M1 L307K, P80C, A160CL307K, P80C, A160C 61.6 61.6 3030 Hy1-A4-M2Hy1-A4-M2 L307K, G291C, Y362CL307K, G291C, Y362C 60.7 60.7 3131 Hy1-A4-M3Hy1-A4-M3 L307K, L180C, A223CL307K, L180C, A223C 61.5 61.5 3232 Hy1-A4-M4Hy1-A4-M4 L307K, P6C, Y403CL307K, P6C, Y403C 60.6 60.6 3333 Hy1-A4-M5Hy1-A4-M5 L307K, S36C, D426CL307K, S36C, D426C 61.1 61.1 3434 Hy1-A5Hy1-A5 T306AT306A 61.6 61.6 3535 Hy1-A5-M1Hy1-A5-M1 T306A, P80C, A160CT306A, P80C, A160C 62.5 62.5 3636 Hy1-A5-M2Hy1-A5-M2 T306A, G291C, Y362CT306A, G291C, Y362C 61.6 61.6 3737 Hy1-A5-M3Hy1-A5-M3 T306A, L180C, A223CT306A, L180C, A223C 62.5 62.5 3838 Hy1-A5-M4Hy1-A5-M4 T306A, P6C, Y403CT306A, P6C, Y403C 61.6 61.6 3939 Hy1-A5-M5Hy1-A5-M5 T306A, S36C, D426CT306A, S36C, D426C 62.0 62.0 4040 Hy2-M1Hy2-M1 P80C, A160CP80C, A160C 63.1 63.1 4141 Hy2-M2Hy2-M2 G291C, Y362CG291C, Y362C 62.2 62.2 4242 Hy2-M3Hy2-M3 L180C, A223CL180C, A223C 63.0 63.0 4343 Hy2-M4Hy2-M4 P6C, Y403CP6C, Y403C 62.1 62.1 4444 Hy2-M5Hy2-M5 S36C, D426CS36C, D426C 62.6 62.6 4545 Hy2-A1Hy2-A1 Y57HY57H 62.2 62.2 4646 Hy2-A1-M1Hy2-A1-M1 Y57H, P80C, A160CY57H, P80C, A160C 63.1 63.1 4747 Hy2-A1-M2Hy2-A1-M2 Y57H, G291C, Y362CY57H, G291C, Y362C 62.2 62.2 4848 Hy2-A1-M3Hy2-A1-M3 Y57H, L180C, A223CY57H, L180C, A223C 63.1 63.1 4949 Hy2-A1-M4Hy2-A1-M4 Y57H, P6C, Y403CY57H, P6C, Y403C 62.2 62.2 5050 Hy2-A1-M5Hy2-A1-M5 Y57H, S36C, D426CY57H, S36C, D426C 62.6 62.6 5151 Hy2-A2Hy2-A2 W304HW304H 62.2 62.2 5252 Hy2-A2-M1Hy2-A2-M1 W304H, P80C, A160CW304H, P80C, A160C 63.1 63.1 5353 Hy2-A2-M2Hy2-A2-M2 W304H, G291C, Y362CW304H, G291C, Y362C 62.2 62.2 5454 Hy2-A2-M3Hy2-A2-M3 W304H, L180C, A223CW304H, L180C, A223C 63.0 63.0 5555 Hy2-A2-M4Hy2-A2-M4 W304H, P6C, Y403CW304H, P6C, Y403C 62.2 62.2 5656 Hy2-A2-M5Hy2-A2-M5 W304H, S36C, D426CW304H, S36C, D426C 62.6 62.6 5757 Hy2-A3Hy2-A3 T306RT306R 62.5 62.5 5858 Hy2-A3-M1Hy2-A3-M1 T306R, P80C, A160CT306R, P80C, A160C 63.4 63.4 5959 Hy2-A3-M2Hy2-A3-M2 T306R, G291C, Y362CT306R, G291C, Y362C 62.5 62.5 6060 Hy2-A3-M3Hy2-A3-M3 T306R, L180C, A223CT306R, L180C, A223C 63.4 63.4 6161 Hy2-A3-M4Hy2-A3-M4 T306R, P6C, Y403CT306R, P6C, Y403C 62.5 62.5 6262 Hy2-A3-M5Hy2-A3-M5 T306R, S36C, D426CT306R, S36C, D426C 62.9 62.9 6363 Hy2-A4Hy2-A4 L307KL307K 61.3 61.3 6464 Hy2-A4-M1Hy2-A4-M1 L307K, P80C, A160CL307K, P80C, A160C 62.2 62.2 6565 Hy2-A4-M2Hy2-A4-M2 L307K, G291C, Y362CL307K, G291C, Y362C 61.3 61.3 6666 Hy2-A4-M3Hy2-A4-M3 L307K, L180C, A223CL307K, L180C, A223C 62.2 62.2 6767 Hy2-A4-M4Hy2-A4-M4 L307K, P6C, Y403CL307K, P6C, Y403C 61.3 61.3 6868 Hy2-A4-M5Hy2-A4-M5 L307K, S36C, D426CL307K, S36C, D426C 61.8 61.8 6969 Hy2-A5Hy2-A5 T306AT306A 62.3 62.3 7070 Hy2-A5-M1Hy2-A5-M1 T306A, P80C, A160CT306A, P80C, A160C 63.2 63.2 7171 Hy2-A5-M2Hy2-A5-M2 T306A, G291C, Y362CT306A, G291C, Y362C 62.3 62.3 7272 Hy2-A5-M3Hy2-A5-M3 T306A, L180C, A223CT306A, L180C, A223C 63.2 63.2 7373 Hy2-A5-M4Hy2-A5-M4 T306A, P6C, Y403CT306A, P6C, Y403C 62.3 62.3 7474 Hy2-A5-M5Hy2-A5-M5 T306A, S36C, D426CT306A, S36C, D426C 62.7 62.7 7575 Hy3-M1Hy3-M1 P80C, A160CP80C, A160C 63.5 63.5 7676 Hy3-M2Hy3-M2 G291C, Y362CG291C, Y362C 62.6 62.6 7777 Hy3-M3Hy3-M3 L180C, A223CL180C, A223C 63.5 63.5 7878 Hy3-M4Hy3-M4 P6C, Y403CP6C, Y403C 62.6 62.6 7979 Hy3-M5Hy3-M5 S36C, D426CS36C, D426C 63.0 63.0 8080 Hy3-A1Hy3-A1 Y57HY57H 62.6 62.6 8181 Hy3-A1-M1Hy3-A1-M1 Y57H, P80C, A160CY57H, P80C, A160C 63.6 63.6 8282 Hy3-A1-M2Hy3-A1-M2 Y57H, G291C, Y362CY57H, G291C, Y362C 62.7 62.7 8383 Hy3-A1-M3Hy3-A1-M3 Y57H, L180C, A223CY57H, L180C, A223C 63.5 63.5 8484 Hy3-A1-M4Hy3-A1-M4 Y57H, P6C, Y403CY57H, P6C, Y403C 62.6 62.6 8585 Hy3-A1-M5Hy3-A1-M5 Y57H, S36C, D426CY57H, S36C, D426C 63.1 63.1 8686 Hy3-A2Hy3-A2 W304HW304H 62.6 62.6 8787 Hy3-A2-M1Hy3-A2-M1 W304H, P80C, A160CW304H, P80C, A160C 63.5 63.5 8888 Hy3-A2-M2Hy3-A2-M2 W304H, G291C, Y362CW304H, G291C, Y362C 62.6 62.6 8989 Hy3-A2-M3Hy3-A2-M3 W304H, L180C, A223CW304H, L180C, A223C 63.5 63.5 9090 Hy3-A2-M4Hy3-A2-M4 W304H, P6C, Y403CW304H, P6C, Y403C 62.6 62.6 9191 Hy3-A2-M5Hy3-A2-M5 W304H, S36C, D426CW304H, S36C, D426C 63.0 63.0 9292 Hy3-A3Hy3-A3 T306RT306R 62.9 62.9 9393 Hy3-A3-M1Hy3-A3-M1 T306R, P80C, A160CT306R, P80C, A160C 63.9 63.9 9494 Hy3-A3-M2Hy3-A3-M2 T306R, G291C, Y362CT306R, G291C, Y362C 63.0 63.0 9595 Hy3-A3-M3Hy3-A3-M3 T306R, L180C, A223CT306R, L180C, A223C 63.8 63.8 9696 Hy3-A3-M4Hy3-A3-M4 T306R, P6C, Y403CT306R, P6C, Y403C 62.9 62.9 9797 Hy3-A3-M5Hy3-A3-M5 T306R, S36C, D426CT306R, S36C, D426C 63.4 63.4 9898 Hy3-A4Hy3-A4 L307KL307K 61.8 61.8 9999 Hy3-A4-M1Hy3-A4-M1 L307K, P80C, A160CL307K, P80C, A160C 62.7 62.7 100100 Hy3-A4-M2Hy3-A4-M2 L307K, G291C, Y362CL307K, G291C, Y362C 61.8 61.8 101101 Hy3-A4-M3Hy3-A4-M3 L307K, L180C, A223CL307K, L180C, A223C 62.7 62.7 102102 Hy3-A4-M4Hy3-A4-M4 L307K, P6C, Y403CL307K, P6C, Y403C 61.8 61.8 103103 Hy3-A4-M5Hy3-A4-M5 L307K, S36C, D426CL307K, S36C, D426C 62.2 62.2 104104 Hy3-A5Hy3-A5 T306AT306A 62.7 62.7 105105 Hy3-A5-M1Hy3-A5-M1 T306A, P80C, A160CT306A, P80C, A160C 63.6 63.6 106106 Hy3-A5-M2Hy3-A5-M2 T306A, G291C, Y362CT306A, G291C, Y362C 62.7 62.7 107107 Hy3-A5-M3Hy3-A5-M3 T306A, L180C, A223CT306A, L180C, A223C 63.6 63.6 108108 Hy3-A5-M4Hy3-A5-M4 T306A, P6C, Y403CT306A, P6C, Y403C 62.7 62.7 109109 Hy3-A5-M5Hy3-A5-M5 T306A, S36C, D426CT306A, S36C, D426C 63.2 63.2

실시예 5. 히알루로니다제 PH-20 변이체 안정성 평가 (37℃, 3 days)Example 5. Stability evaluation of hyaluronidase PH-20 variant (37°C, 3 days)

Active site의 변이 또는 시스테인을 도입하여 내부에 신규한 이황화결합을 유도한 히알루로니다제 PH-20 변이체의 수용액 안정성을 확인하기 위하여, 37℃ incubation test를 진행하였다. 인체의 상태와 가장 유사한 PBS (phosphate buffer saline) 상태에서 야생형 PH-20과 변이체들을 0.5 mg/ml로 녹인 후 37℃ water bath에서 incubation을 한 다음, 1일 단위로 sampling 하여 13000 rpm으로 15분간 4℃에서 원심 분리하여 상층액만 얻어 효소활성평가를 수행하였다.To confirm the aqueous solution stability of the hyaluronidase PH-20 variant, which induced a new internal disulfide bond by mutation of the active site or introduction of cysteine, a 37°C incubation test was performed. Wild-type PH-20 and its variants were dissolved at 0.5 mg/ml in PBS (phosphate buffer saline), which is most similar to the state of the human body, and then incubated in a 37°C water bath, then sampled daily and incubated at 13,000 rpm for 15 minutes. Enzyme activity evaluation was performed by centrifuging at ℃ to obtain only the supernatant.

그 결과, 히알루로니다제의 37℃에서 안정성은 하기의 표 5와 같았으며, 그 중 일부에 대하여 도 4에 도시하였다.As a result, the stability of hyaluronidase at 37°C was as shown in Table 5 below, and some of them are shown in Figure 4.

안정성 평가 (37℃, days)Stability evaluation (37℃, days) 서열번호sequence number 명칭designation 00 1One 22 33 22 Hy1Hy1 100100 80.4880.48 50.8150.81 25.2125.21 33 Hy2Hy2 100100 87.6987.69 61.0161.01 36.5036.50 44 Hy3Hy3 100100 89.2889.28 62.5162.51 41.6541.65 55 Hy1-M1Hy1-M1 100100 85.6185.61 72.6572.65 39.6639.66 66 Hy1-M2Hy1-M2 100100 85.2885.28 56.5156.51 25.7625.76 77 Hy1-M3Hy1-M3 100100 82.6882.68 68.0768.07 39.4239.42 88 Hy1-M4Hy1-M4 100100 83.4483.44 56.8756.87 24.8424.84 99 Hy1-M5Hy1-M5 100100 83.6983.69 68.7268.72 34.7534.75 1010 Hy1-A1Hy1-A1 100100 82.0982.09 54.3154.31 27.3127.31 1111 Hy1-A1-M1Hy1-A1-M1 100100 83.6783.67 55.3955.39 40.4740.47 1212 Hy1-A1-M2Hy1-A1-M2 100100 87.3487.34 61.0061.00 27.8227.82 1313 Hy1-A1-M3Hy1-A1-M3 100100 86.7886.78 76.5376.53 40.0140.01 1414 Hy1-A1-M4Hy1-A1-M4 100100 80.4180.41 53.7053.70 27.1427.14 1515 Hy1-A1-M5Hy1-A1-M5 100100 80.7380.73 54.0754.07 35.1035.10 1616 Hy1-A2Hy1-A2 100100 83.9183.91 57.1557.15 25.7325.73 1717 Hy1-A2-M1Hy1-A2-M1 100100 86.5286.52 64.7664.76 40.2140.21 1818 Hy1-A2-M2Hy1-A2-M2 100100 89.0589.05 66.3266.32 26.7726.77 1919 Hy1-A2-M3Hy1-A2-M3 100100 84.4584.45 66.2566.25 39.6039.60 2020 Hy1-A2-M4Hy1-A2-M4 100100 85.0685.06 58.8758.87 25.4225.42 2121 Hy1-A2-M5Hy1-A2-M5 100100 86.0786.07 66.7766.77 35.0635.06 2222 Hy1-A3Hy1-A3 100100 87.2287.22 69.5069.50 34.0734.07 2323 Hy1-A3-M1Hy1-A3-M1 100100 88.9788.97 67.8467.84 45.6145.61 2424 Hy1-A3-M2Hy1-A3-M2 100100 85.6385.63 70.2570.25 34.4634.46 2525 Hy1-A3-M3Hy1-A3-M3 100100 87.6987.69 73.8873.88 44.0444.04 2626 Hy1-A3-M4Hy1-A3-M4 100100 84.3184.31 62.7062.70 34.0034.00 2727 Hy1-A3-M5Hy1-A3-M5 100100 80.8580.85 58.2658.26 38.4938.49 2828 Hy1-A4Hy1-A4 100100 80.5580.55 54.9854.98 21.1221.12 2929 Hy1-A4-M1Hy1-A4-M1 100100 89.5089.50 65.6165.61 28.0928.09 3030 Hy1-A4-M2Hy1-A4-M2 100100 86.3986.39 61.5661.56 22.7822.78 3131 Hy1-A4-M3Hy1-A4-M3 100100 83.8783.87 58.7558.75 27.5527.55 3232 Hy1-A4-M4Hy1-A4-M4 100100 83.5783.57 56.0856.08 18.1018.10 3333 Hy1-A4-M5Hy1-A4-M5 100100 80.3580.35 52.7052.70 23.3523.35 3434 Hy1-A5Hy1-A5 100100 81.8981.89 68.8968.89 29.3629.36 3535 Hy1-A5-M1Hy1-A5-M1 100100 88.4188.41 69.8269.82 41.5541.55 3636 Hy1-A5-M2Hy1-A5-M2 100100 81.8981.89 54.5054.50 30.0630.06 3737 Hy1-A5-M3Hy1-A5-M3 100100 82.4382.43 66.5066.50 40.5140.51 3838 Hy1-A5-M4Hy1-A5-M4 100100 85.3785.37 64.8364.83 29.0729.07 3939 Hy1-A5-M5Hy1-A5-M5 100100 82.0182.01 52.8552.85 35.1435.14 4040 Hy2-M1Hy2-M1 100100 88.0088.00 58.8958.89 48.2148.21 4141 Hy2-M2Hy2-M2 100100 82.7882.78 66.8066.80 36.8136.81 4242 Hy2-M3Hy2-M3 100100 86.3986.39 68.6168.61 46.7846.78 4343 Hy2-M4Hy2-M4 100100 82.5382.53 71.4071.40 36.4536.45 4444 Hy2-M5Hy2-M5 100100 83.8383.83 70.4870.48 41.7841.78 4545 Hy2-A1Hy2-A1 100100 81.6181.61 67.5767.57 37.1237.12 4646 Hy2-A1-M1Hy2-A1-M1 100100 81.5881.58 68.2868.28 49.4649.46 4747 Hy2-A1-M2Hy2-A1-M2 100100 86.5386.53 67.2367.23 37.4437.44 4848 Hy2-A1-M3Hy2-A1-M3 100100 83.6083.60 73.3673.36 49.2049.20 4949 Hy2-A1-M4Hy2-A1-M4 100100 80.0780.07 65.7565.75 37.2737.27 5050 Hy2-A1-M5Hy2-A1-M5 100100 86.6786.67 67.0267.02 43.4343.43 5151 Hy2-A2Hy2-A2 100100 86.0786.07 61.6561.65 36.6836.68 5252 Hy2-A2-M1Hy2-A2-M1 100100 87.0487.04 65.5065.50 49.1249.12 5353 Hy2-A2-M2Hy2-A2-M2 100100 84.5084.50 74.0374.03 36.9136.91 5454 Hy2-A2-M3Hy2-A2-M3 100100 80.4280.42 70.0770.07 47.4247.42 5555 Hy2-A2-M4Hy2-A2-M4 100100 86.1086.10 59.6359.63 36.5536.55 5656 Hy2-A2-M5Hy2-A2-M5 100100 86.4286.42 72.8972.89 42.7542.75 5757 Hy2-A3Hy2-A3 100100 88.0088.00 58.8158.81 41.1341.13 5858 Hy2-A3-M1Hy2-A3-M1 100100 85.9185.91 71.9971.99 54.3754.37 5959 Hy2-A3-M2Hy2-A3-M2 100100 81.0181.01 65.9765.97 41.5241.52 6060 Hy2-A3-M3Hy2-A3-M3 100100 83.0583.05 72.6172.61 53.4553.45 6161 Hy2-A3-M4Hy2-A3-M4 100100 86.8886.88 57.8657.86 40.8340.83 6262 Hy2-A3-M5Hy2-A3-M5 100100 83.7583.75 58.0758.07 46.6746.67 6363 Hy2-A4Hy2-A4 100100 81.1781.17 57.2657.26 24.2124.21 6464 Hy2-A4-M1Hy2-A4-M1 100100 81.6781.67 56.2056.20 37.4937.49 6565 Hy2-A4-M2Hy2-A4-M2 100100 88.8088.80 61.0161.01 24.3224.32 6666 Hy2-A4-M3Hy2-A4-M3 100100 87.5287.52 70.6170.61 37.3837.38 6767 Hy2-A4-M4Hy2-A4-M4 100100 84.9984.99 62.1262.12 24.0524.05 6868 Hy2-A4-M5Hy2-A4-M5 100100 83.8683.86 71.2671.26 32.0832.08 6969 Hy2-A5Hy2-A5 100100 84.6084.60 63.2163.21 37.7537.75 7070 Hy2-A5-M1Hy2-A5-M1 100100 88.2588.25 75.6875.68 50.7150.71 7171 Hy2-A5-M2Hy2-A5-M2 100100 88.2588.25 60.2560.25 39.0439.04 7272 Hy2-A5-M3Hy2-A5-M3 100100 80.7580.75 65.9065.90 50.4950.49 7373 Hy2-A5-M4Hy2-A5-M4 100100 80.7280.72 60.2760.27 37.6037.60 7474 Hy2-A5-M5Hy2-A5-M5 100100 85.6485.64 60.3260.32 44.9544.95 7575 Hy3-M1Hy3-M1 100100 89.7389.73 67.5067.50 56.2456.24 7676 Hy3-M2Hy3-M2 100100 86.2286.22 67.8067.80 42.9042.90 7777 Hy3-M3Hy3-M3 100100 84.1884.18 71.0871.08 55.6055.60 7878 Hy3-M4Hy3-M4 100100 85.2485.24 68.6568.65 41.6541.65 7979 Hy3-M5Hy3-M5 100100 81.9081.90 59.1459.14 47.0747.07 8080 Hy3-A1Hy3-A1 100100 84.8284.82 56.6756.67 43.3643.36 8181 Hy3-A1-M1Hy3-A1-M1 100100 85.4385.43 74.0974.09 57.9257.92 8282 Hy3-A1-M2Hy3-A1-M2 100100 86.1386.13 75.3775.37 43.8643.86 8383 Hy3-A1-M3Hy3-A1-M3 100100 87.1987.19 73.1873.18 56.5656.56 8484 Hy3-A1-M4Hy3-A1-M4 100100 81.0681.06 69.6269.62 43.3043.30 8585 Hy3-A1-M5Hy3-A1-M5 100100 85.6185.61 67.0567.05 48.6448.64 8686 Hy3-A2Hy3-A2 100100 83.7783.77 73.2873.28 42.5042.50 8787 Hy3-A2-M1Hy3-A2-M1 100100 84.7384.73 68.0468.04 57.5657.56 8888 Hy3-A2-M2Hy3-A2-M2 100100 81.9581.95 56.9356.93 43.1343.13 8989 Hy3-A2-M3Hy3-A2-M3 100100 84.2884.28 71.4971.49 56.1156.11 9090 Hy3-A2-M4Hy3-A2-M4 100100 81.8881.88 63.1263.12 41.9041.90 9191 Hy3-A2-M5Hy3-A2-M5 100100 80.8480.84 59.7959.79 47.8047.80 9292 Hy3-A3Hy3-A3 100100 86.6886.68 74.1174.11 46.5546.55 9393 Hy3-A3-M1Hy3-A3-M1 100100 84.7584.75 74.2374.23 62.1962.19 9494 Hy3-A3-M2Hy3-A3-M2 100100 81.8981.89 58.0458.04 46.6946.69 9595 Hy3-A3-M3Hy3-A3-M3 100100 84.7084.70 72.6172.61 60.9860.98 9696 Hy3-A3-M4Hy3-A3-M4 100100 89.1789.17 70.9470.94 45.8345.83 9797 Hy3-A3-M5Hy3-A3-M5 100100 83.2683.26 70.3970.39 53.0253.02 9898 Hy3-A4Hy3-A4 100100 81.3581.35 53.1653.16 31.9631.96 9999 Hy3-A4-M1Hy3-A4-M1 100100 88.8188.81 72.7672.76 43.9243.92 100100 Hy3-A4-M2Hy3-A4-M2 100100 80.8580.85 67.8767.87 33.1233.12 101101 Hy3-A4-M3Hy3-A4-M3 100100 83.6083.60 70.6470.64 43.7043.70 102102 Hy3-A4-M4Hy3-A4-M4 100100 82.3582.35 54.6354.63 30.6830.68 103103 Hy3-A4-M5Hy3-A4-M5 100100 89.3289.32 72.5472.54 37.3437.34 104104 Hy3-A5Hy3-A5 100100 88.5388.53 59.9159.91 44.8244.82 105105 Hy3-A5-M1Hy3-A5-M1 100100 84.0984.09 72.3072.30 59.2159.21 106106 Hy3-A5-M2Hy3-A5-M2 100100 83.2883.28 66.2566.25 45.5345.53 107107 Hy3-A5-M3Hy3-A5-M3 100100 80.5280.52 70.1070.10 58.9558.95 108108 Hy3-A5-M4Hy3-A5-M4 100100 82.6482.64 72.1072.10 44.3344.33 109109 Hy3-A5-M5Hy3-A5-M5 100100 84.2084.20 60.8860.88 50.3650.36

실시예 6. 히알루로니다제 PH-20 변이체 안정성 평가 (50℃, 3 hours)Example 6. Stability evaluation of hyaluronidase PH-20 variant (50°C, 3 hours)

Active site의 변이 또는 시스테인을 도입하여 내부에 신규한 이황화결합을 유도한 히알루로니다제 PH-20 변이체의 수용액 안정성을 확인하기 위하여, 50℃ incubation test를 진행하였다. 인체의 상태와 가장 유사한 PBS (phosphate buffer saline) 상태에서 야생형 PH-20과 변이체들을 0.5 mg/ml로 녹인 후 50℃ water bath에서 incubation을 한 다음, 1시간 단위로 sampling 하여 13000 rpm으로 15분간 4℃에서 원심 분리하여 상층액만 얻어 효소활성평가를 수행하였다. To confirm the aqueous solution stability of the hyaluronidase PH-20 variant, which induced a new disulfide bond internally by mutation of the active site or introduction of cysteine, a 50°C incubation test was performed. Wild-type PH-20 and its variants were dissolved at 0.5 mg/ml in PBS (phosphate buffer saline), which is most similar to the state of the human body, and then incubated in a 50°C water bath, followed by sampling every hour and incubation at 13,000 rpm for 15 minutes. Enzyme activity evaluation was performed by centrifuging at ℃ to obtain only the supernatant.

그 결과, 히알루로니다제의 50℃에서 안정성은 하기의 표 6과 같았으며, 그 중 일부에 대하여 도 5에 도시하였다.As a result, the stability of hyaluronidase at 50°C was as shown in Table 6 below, and some of them are shown in Figure 5.

안정성 평가 (50℃, hours)Stability evaluation (50℃, hours) 서열번호sequence number 명칭designation 00 1One 22 33 22 Hy1Hy1 100100 77.1977.19 22.4622.46 1.021.02 33 Hy2Hy2 100100 75.6875.68 31.2931.29 5.165.16 44 Hy3Hy3 100100 78.6278.62 34.8734.87 10.9110.91 55 Hy1-M1Hy1-M1 100100 68.3168.31 17.3317.33 9.449.44 66 Hy1-M2Hy1-M2 100100 69.7969.79 22.4522.45 2.152.15 77 Hy1-M3Hy1-M3 100100 64.2864.28 35.2935.29 8.878.87 88 Hy1-M4Hy1-M4 100100 63.2563.25 28.8328.83 1.011.01 99 Hy1-M5Hy1-M5 100100 75.0675.06 26.3126.31 4.324.32 1010 Hy1-A1Hy1-A1 100100 60.7260.72 28.3928.39 2.292.29 1111 Hy1-A1-M1Hy1-A1-M1 100100 78.9978.99 30.7130.71 9.829.82 1212 Hy1-A1-M2Hy1-A1-M2 100100 77.4877.48 28.3128.31 2.322.32 1313 Hy1-A1-M3Hy1-A1-M3 100100 72.5872.58 29.5929.59 9.579.57 1414 Hy1-A1-M4Hy1-A1-M4 100100 75.3175.31 27.4827.48 2.282.28 1515 Hy1-A1-M5Hy1-A1-M5 100100 72.7472.74 21.1621.16 4.654.65 1616 Hy1-A2Hy1-A2 100100 75.1175.11 22.4222.42 1.781.78 1717 Hy1-A2-M1Hy1-A2-M1 100100 67.2467.24 32.8832.88 9.599.59 1818 Hy1-A2-M2Hy1-A2-M2 100100 79.8579.85 29.7529.75 2.222.22 1919 Hy1-A2-M3Hy1-A2-M3 100100 79.3679.36 33.9033.90 9.059.05 2020 Hy1-A2-M4Hy1-A2-M4 100100 62.4862.48 26.6426.64 1.681.68 2121 Hy1-A2-M5Hy1-A2-M5 100100 62.7762.77 29.0929.09 4.564.56 2222 Hy1-A3Hy1-A3 100100 73.1973.19 30.7930.79 4.214.21 2323 Hy1-A3-M1Hy1-A3-M1 100100 64.2864.28 10.5510.55 6.126.12 2424 Hy1-A3-M2Hy1-A3-M2 100100 74.9574.95 17.1417.14 4.244.24 2525 Hy1-A3-M3Hy1-A3-M3 100100 65.1065.10 36.7236.72 14.6114.61 2626 Hy1-A3-M4Hy1-A3-M4 100100 69.8269.82 30.1830.18 4.184.18 2727 Hy1-A3-M5Hy1-A3-M5 100100 64.4764.47 20.4220.42 7.477.47 2828 Hy1-A4Hy1-A4 100100 68.0268.02 28.8828.88 0.240.24 2929 Hy1-A4-M1Hy1-A4-M1 100100 69.6869.68 29.1129.11 2.452.45 3030 Hy1-A4-M2Hy1-A4-M2 100100 78.4178.41 26.1126.11 0.270.27 3131 Hy1-A4-M3Hy1-A4-M3 100100 73.9373.93 20.8320.83 2.292.29 3232 Hy1-A4-M4Hy1-A4-M4 100100 66.4266.42 22.0622.06 0.090.09 3333 Hy1-A4-M5Hy1-A4-M5 100100 62.0562.05 29.8729.87 0.420.42 3434 Hy1-A5Hy1-A5 100100 70.5970.59 22.3522.35 3.043.04 3535 Hy1-A5-M1Hy1-A5-M1 100100 76.6876.68 33.6633.66 10.8410.84 3636 Hy1-A5-M2Hy1-A5-M2 100100 64.6964.69 20.5220.52 3.163.16 3737 Hy1-A5-M3Hy1-A5-M3 100100 78.7878.78 30.0030.00 9.869.86 3838 Hy1-A5-M4Hy1-A5-M4 100100 60.2460.24 24.3024.30 3.013.01 3939 Hy1-A5-M5Hy1-A5-M5 100100 69.5369.53 27.0827.08 4.884.88 4040 Hy2-M1Hy2-M1 100100 71.4071.40 39.4539.45 18.6318.63 4141 Hy2-M2Hy2-M2 100100 65.0965.09 20.4920.49 5.965.96 4242 Hy2-M3Hy2-M3 100100 72.0472.04 43.9943.99 17.8417.84 4343 Hy2-M4Hy2-M4 100100 74.5074.50 17.3617.36 5.135.13 4444 Hy2-M5Hy2-M5 100100 63.6063.60 12.6212.62 11.0911.09 4545 Hy2-A1Hy2-A1 100100 60.8460.84 28.7028.70 6.026.02 4646 Hy2-A1-M1Hy2-A1-M1 100100 79.5579.55 50.0950.09 21.0021.00 4747 Hy2-A1-M2Hy2-A1-M2 100100 68.2368.23 35.9935.99 6.416.41 4848 Hy2-A1-M3Hy2-A1-M3 100100 66.8766.87 9.509.50 0.020.02 4949 Hy2-A1-M4Hy2-A1-M4 100100 63.0663.06 28.9128.91 6.226.22 5050 Hy2-A1-M5Hy2-A1-M5 100100 71.7171.71 43.9543.95 14.0114.01 5151 Hy2-A2Hy2-A2 100100 71.1871.18 31.1931.19 5.615.61 5252 Hy2-A2-M1Hy2-A2-M1 100100 76.8276.82 48.0048.00 19.8319.83 5353 Hy2-A2-M2Hy2-A2-M2 100100 70.0370.03 20.9320.93 5.975.97 5454 Hy2-A2-M3Hy2-A2-M3 100100 63.4763.47 41.0541.05 18.2618.26 5555 Hy2-A2-M4Hy2-A2-M4 100100 70.2470.24 22.0322.03 5.595.59 5656 Hy2-A2-M5Hy2-A2-M5 100100 63.1663.16 16.6116.61 11.9111.91 5757 Hy2-A3Hy2-A3 100100 60.3560.35 36.7136.71 9.979.97 5858 Hy2-A3-M1Hy2-A3-M1 100100 76.2076.20 44.9944.99 24.8524.85 5959 Hy2-A3-M2Hy2-A3-M2 100100 76.6276.62 18.7918.79 10.8110.81 6060 Hy2-A3-M3Hy2-A3-M3 100100 69.4169.41 44.9344.93 24.6824.68 6161 Hy2-A3-M4Hy2-A3-M4 100100 60.5060.50 11.3111.31 9.889.88 6262 Hy2-A3-M5Hy2-A3-M5 100100 70.3970.39 11.8011.80 7.287.28 6363 Hy2-A4Hy2-A4 100100 74.7074.70 24.3024.30 0.610.61 6464 Hy2-A4-M1Hy2-A4-M1 100100 64.6364.63 33.2333.23 6.516.51 6565 Hy2-A4-M2Hy2-A4-M2 100100 62.3962.39 25.4225.42 0.720.72 6666 Hy2-A4-M3Hy2-A4-M3 100100 74.5474.54 36.0936.09 6.386.38 6767 Hy2-A4-M4Hy2-A4-M4 100100 74.3374.33 19.8019.80 0.560.56 6868 Hy2-A4-M5Hy2-A4-M5 100100 60.4760.47 19.9719.97 4.114.11 6969 Hy2-A5Hy2-A5 100100 62.6362.63 19.9519.95 7.387.38 7070 Hy2-A5-M1Hy2-A5-M1 100100 76.3076.30 46.3446.34 23.2823.28 7171 Hy2-A5-M2Hy2-A5-M2 100100 62.6462.64 30.8930.89 8.068.06 7272 Hy2-A5-M3Hy2-A5-M3 100100 78.2978.29 45.3745.37 21.4521.45 7373 Hy2-A5-M4Hy2-A5-M4 100100 64.7164.71 34.1734.17 7.037.03 7474 Hy2-A5-M5Hy2-A5-M5 100100 65.3765.37 41.2041.20 15.7615.76 7575 Hy3-M1Hy3-M1 100100 68.0868.08 46.4646.46 25.2025.20 7676 Hy3-M2Hy3-M2 100100 68.4268.42 12.1812.18 11.9911.99 7777 Hy3-M3Hy3-M3 100100 74.8074.80 51.4251.42 24.8824.88 7878 Hy3-M4Hy3-M4 100100 63.5263.52 16.9416.94 10.9010.90 7979 Hy3-M5Hy3-M5 100100 61.2961.29 5.535.53 0.070.07 8080 Hy3-A1Hy3-A1 100100 60.7060.70 38.1638.16 13.6013.60 8181 Hy3-A1-M1Hy3-A1-M1 100100 75.7575.75 54.5254.52 29.6029.60 8282 Hy3-A1-M2Hy3-A1-M2 100100 79.9279.92 34.6834.68 14.1714.17 8383 Hy3-A1-M3Hy3-A1-M3 100100 75.5975.59 46.6746.67 26.4626.46 8484 Hy3-A1-M4Hy3-A1-M4 100100 71.1571.15 35.4735.47 12.7712.77 8585 Hy3-A1-M5Hy3-A1-M5 100100 69.7769.77 45.6845.68 19.3219.32 8686 Hy3-A2Hy3-A2 100100 70.9670.96 34.4534.45 11.4711.47 8787 Hy3-A2-M1Hy3-A2-M1 100100 71.8071.80 49.6749.67 26.5426.54 8888 Hy3-A2-M2Hy3-A2-M2 100100 77.5177.51 34.6634.66 12.0312.03 8989 Hy3-A2-M3Hy3-A2-M3 100100 66.4666.46 45.7745.77 25.1125.11 9090 Hy3-A2-M4Hy3-A2-M4 100100 76.3076.30 34.9934.99 11.2511.25 9191 Hy3-A2-M5Hy3-A2-M5 100100 78.4778.47 41.8241.82 18.6118.61 9292 Hy3-A3Hy3-A3 100100 75.3875.38 38.8638.86 17.1717.17 9393 Hy3-A3-M1Hy3-A3-M1 100100 79.3079.30 57.7757.77 36.7536.75 9494 Hy3-A3-M2Hy3-A3-M2 100100 70.9670.96 43.3743.37 17.5317.53 9595 Hy3-A3-M3Hy3-A3-M3 100100 79.6879.68 57.5257.52 34.7534.75 9696 Hy3-A3-M4Hy3-A3-M4 100100 79.0879.08 44.4244.42 17.1117.11 9797 Hy3-A3-M5Hy3-A3-M5 100100 74.5974.59 47.7747.77 23.4823.48 9898 Hy3-A4Hy3-A4 100100 74.8674.86 18.9918.99 3.743.74 9999 Hy3-A4-M1Hy3-A4-M1 100100 73.7973.79 15.1815.18 14.3414.34 100100 Hy3-A4-M2Hy3-A4-M2 100100 60.7060.70 30.1930.19 4.124.12 101101 Hy3-A4-M3Hy3-A4-M3 100100 63.0663.06 40.0840.08 14.0514.05 102102 Hy3-A4-M4Hy3-A4-M4 100100 72.8372.83 23.3323.33 3.323.32 103103 Hy3-A4-M5Hy3-A4-M5 100100 79.1079.10 29.7429.74 6.316.31 104104 Hy3-A5Hy3-A5 100100 68.8268.82 9.589.58 4.954.95 105105 Hy3-A5-M1Hy3-A5-M1 100100 78.2778.27 53.8653.86 33.0333.03 106106 Hy3-A5-M2Hy3-A5-M2 100100 79.8879.88 45.1745.17 15.9615.96 107107 Hy3-A5-M3Hy3-A5-M3 100100 77.6977.69 55.8555.85 30.3730.37 108108 Hy3-A5-M4Hy3-A5-M4 100100 64.5264.52 42.3542.35 14.6514.65 109109 Hy3-A5-M5Hy3-A5-M5 100100 62.7662.76 6.596.59 1.061.06

이상 살펴본 바와 같이, 본 발명에 따른 신규한 히알루로니다제 PH-20 변이체는 야생형의 인간 히알루로니다제 PH-20와 비교하여 안정성이 높고 활성이 뛰어나므로, 이와 함께 사용되는 약물의 치료 효과를 극대화할 수 있어, 산업상 이용가능성이 높다.As discussed above, the novel hyaluronidase PH-20 variant according to the present invention has high stability and excellent activity compared to wild-type human hyaluronidase PH-20, thereby improving the therapeutic effect of drugs used together with it. Since it can be maximized, it has high industrial applicability.

Claims (22)

서열번호 2, 서열번호 3, 및 서열번호 4으로 이루어진 군에서 선택된 어느 하나의 히알루로니다제 PH-20 변이체.
Any one hyaluronidase PH-20 variant selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.
제1항에 있어서 상기 히알루로니다제 PH-20 변이체는, ⅰ) Y57H, ⅱ) W304H, ⅲ) T306R, ⅵ) L307K, 및 ⅴ) T306A로 이루어진 군에서 선택된 어느 하나의 아미노산 잔기의 치환을 더 포함하는 것을 특징으로 하는 히알루로니다제 PH-20 변이체.
The method of claim 1, wherein the hyaluronidase PH-20 variant further comprises the substitution of any one amino acid residue selected from the group consisting of i) Y57H, ii) W304H, iii) T306R, vi) L307K, and v) T306A. A hyaluronidase PH-20 variant comprising:
제1항에 있어서 상기 히알루로니다제 PH-20 변이체는, ⅰ) Y57H, ⅱ) W304H, ⅲ) T306R, 및 ⅵ) L307K로 이루어진 군에서 선택된 어느 하나의 아미노산 잔기의 치환을 더 포함하는 것을 특징으로 하는 히알루로니다제 PH-20 변이체.
The method of claim 1, wherein the hyaluronidase PH-20 variant further comprises a substitution of any one amino acid residue selected from the group consisting of i) Y57H, ii) W304H, iii) T306R, and vi) L307K. Hyaluronidase PH-20 variant.
제1항에 있어서 상기 히알루로니다제 PH-20 변이체는 ⅰ) P80C 및 A160C, ⅱ) G291C 및 Y362C, ⅲ) L180C 및 A223C, ⅵ) P6C 및 Y403C, 및 ⅴ) S36C 및 D426C로 이루어진 군에서 선택된 어느 하나의 아미노산 잔기의 치환을 더 포함하는 것을 특징으로 하는 히알루로니다제 PH-20 변이체.
The method of claim 1, wherein the hyaluronidase PH-20 variant is i) P80C and A160C, ii) G291C and Y362C, iii) L180C and A223C, vi) P6C and Y403C, and v) a hyaluronidase PH-20 variant, characterized in that it further comprises the substitution of any one amino acid residue selected from the group consisting of S36C and D426C.
제1항에 있어서 상기 히알루로니다제 PH-20 변이체는 ⅰ) G291C 및 Y362C, ⅱ) L180C 및 A223C, ⅲ) P6C 및 Y403C, 및 ⅵ) S36C 및 D426C로 이루어진 군에서 선택된 어느 하나의 아미노산 잔기의 치환을 더 포함하는 것을 특징으로 하는 히알루로니다제 PH-20 변이체.
The method of claim 1, wherein the hyaluronidase PH-20 variant is one amino acid residue selected from the group consisting of i) G291C and Y362C, ii) L180C and A223C, iii) P6C and Y403C, and vi) S36C and D426C. A hyaluronidase PH-20 variant characterized by further comprising substitutions.
제2항에 있어서 상기 히알루로니다제 PH-20 변이체는 ⅰ) P80C 및 A160C, ⅱ) G291C 및 Y362C, ⅲ) L180C 및 A223C, ⅵ) P6C 및 Y403C, 및 ⅴ) S36C 및 D426C로 이루어진 군에서 선택된 어느 하나의 아미노산 잔기의 치환을 더 포함하는 것을 특징으로 하는 히알루로니다제 PH-20 변이체.
The method of claim 2, wherein the hyaluronidase PH-20 variant is selected from the group consisting of i) P80C and A160C, ii) G291C and Y362C, iii) L180C and A223C, vi) P6C and Y403C, and v) S36C and D426C. Hyaluronidase PH-20 variant, characterized in that it further comprises a substitution of any one amino acid residue.
제1항 내지 제6항 중 어느 한 항의 히알루로니다제 PH-20 변이체를 암호화하는 폴리뉴클레오티드.
A polynucleotide encoding the hyaluronidase PH-20 variant of any one of claims 1 to 6.
제7항의 폴리뉴클레오티드를 포함하는 발현 벡터.
An expression vector containing the polynucleotide of claim 7.
제8항의 발현 벡터를 포함하는 숙주 세포주.
A host cell line containing the expression vector of claim 8.
제9항에 따른 숙주 세포주를 배양하는 단계를 포함하는 히알루로니다제 PH-20 변이체의 제조 방법.
A method for producing a hyaluronidase PH-20 variant comprising culturing the host cell line according to claim 9.
a) 약물; 및
b) 제1항 내지 제6항 중 어느 한 항의 히알루로니다제 PH-20 변이체;
를 포함하는 것을 특징으로 하는 질환의 예방 또는 치료용 약학적 조성물.
a) drugs; and
b) the hyaluronidase PH-20 variant of any one of claims 1 to 6;
A pharmaceutical composition for preventing or treating diseases, comprising:
제11항에 있어서, 상기 약물은 단백질 의약품(protein drugs), 소분자 화합물(small molecules), 앱타머(aptamer), RNAi, antisense 또는 세포 치료제인 것을 특징으로 하는 약학적 조성물.
The pharmaceutical composition according to claim 11, wherein the drug is a protein drug, small molecule compound, aptamer, RNAi, antisense, or cell therapy.
제11항에 있어서, 상기 약물은 화학요법제, 진통제, 항염증제, 항미생물제, 살구균제, 살트리코모나스제, 항파킨슨제, 항말라리아제, 항경련제, 항우울제, 항관졀염제, 항진균제, 항고혈압제, 해열제, 항기생충제, 항히스타민제, 알파-아드레날린 작용제, 알파 차단제, 마취제, 기관지 확장제, 살균제, 정균제, 베타-아드레날린성 차단제, 칼슘 통로 차단제, 심혈관계 약물, 피임제, 충혈 완화제, 이뇨제, 진정제, 진단제, 전해질제, 최면제, 호르몬제, 혈당 강하제, 근육이완제, 근육수축제, 안과제, 부교감신경 흥분제, 교감신경 흥분제, 살바이러스제, 비타민제, 비스테로이드성 소염제, 안지오텐신 전환효소 억제제, 및 수면유도제로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 약학적 조성물.
The method of claim 11, wherein the drug is a chemotherapy agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial agent, a bactericidal agent, a Saltrichomonas agent, an anti-Parkinsonian agent, an antimalarial agent, an anticonvulsant, an antidepressant, an anti-inflammatory agent, an antifungal agent, an antihypertensive agent, an antipyretic, Antiparasitic agents, antihistamines, alpha-adrenergic agonists, alpha blockers, anesthetics, bronchodilators, bactericides, bacteriostatic agents, beta-adrenergic blockers, calcium channel blockers, cardiovascular drugs, contraceptives, decongestants, diuretics, sedatives, diagnostic agents, A group consisting of electrolytes, hypnotics, hormones, hypoglycemic agents, muscle relaxants, muscle constrictors, ophthalmic agents, parasympathomimetics, sympathomimetics, virucide, vitamins, non-steroidal anti-inflammatory drugs, angiotensin converting enzyme inhibitors, and sleep inducers. A pharmaceutical composition characterized in that it is any one selected from.
제11항에 있어서, 상기 약물은 항체, 수용성 수용체, 또는 수용성 수용체와 Fc 융합 단백질인 것을 특징으로 하는 약학적 조성물.
The pharmaceutical composition according to claim 11, wherein the drug is an antibody, a water-soluble receptor, or a water-soluble receptor and Fc fusion protein.
제14항에 있어서, 상기 항체는 4-1BB, 인테그린 (integrin), 아밀로이드 베타 (amyloid beta), 안지오포에틴, 안지오포에틴 유사물질3, B세포활성인자 (B-cell activating factor, BAFF), B7-H3, 보체5 (complement 5), CCR4, CD3, CD4, CD6, CD11a, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD62, CD79b, CD80, CGRP, 클라우딘18 (Claudin-18), 보체요소 D (complement factor D), CTLA4, DLL3, EGF 수용체, 혈우병인자, Fc 수용체, FGF23, 폴레이트 (folate) 수용체, GD2, GM-CSF, HER2, HER3, 인터페론 수용체, 인터페론 감마, IgE, IGF-1 수용체, 인터루킨1, 인터루킨2 수용체, 인터루킨4 수용체, 인터루킨5, 인터루킨5 수용체, 인터루킨6, 인터루킨6 수용체, 인터루킨7, 인터루킨12/23, 인터루킨13, 인터루킨17A, 인터루킨17 수용체A, 인터루킨31 수용체, 인터루킨36 수용체, LAG3, LFA3, NGF, PVSK9, PD-1, PD-L1, RANK-L, SLAMF7, 조직인자 (Tissue factor), TNF, VEGF, 및 vWF로 이루어진 군에서 선택되는 하나 이상의 항원과 결합하는 것을 특징으로 하는 약학 조성물.
The method of claim 14, wherein the antibody is 4-1BB, integrin, amyloid beta, angiopoietin, angiopoietin-like substance 3, B-cell activating factor (BAFF), B7-H3, complement 5, CCR4, CD3, CD4, CD6, CD11a, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD62, CD79b, CD80, CGRP, Claudin-18 , complement factor D, CTLA4, DLL3, EGF receptor, hemophilia factor, Fc receptor, FGF23, folate receptor, GD2, GM-CSF, HER2, HER3, interferon receptor, interferon gamma, IgE, IGF-1 receptor, interleukin 1, interleukin 2 receptor, interleukin 4 receptor, interleukin 5, interleukin 5 receptor, interleukin 6, interleukin 6 receptor, interleukin 7, interleukin 12/23, interleukin 13, interleukin 17A, interleukin 17 receptor A, interleukin 31 receptor, interleukin 36 receptor, LAG3, LFA3, NGF, PVSK9, PD-1, PD-L1, RANK-L, SLAMF7, tissue factor, TNF, VEGF, and one or more selected from the group consisting of vWF A pharmaceutical composition characterized in that it binds to an antigen.
제14항에 있어서, 상기 항체는 유토밀루맙 (Utomilumab), 나탈리주맙 (Natalizumab), 에트롤리주맙 (Etrolizumab), 베돌리주맙 (Vedolizumab), 비마그루맙 (Bimagrumab), 바피네우주맙 (Bapineuzumab), 크레네주맙(Crenezumab), 솔라네주맙 (Solanezumab), 아두카누맙 (Aducanumab), 간테네루맙 (Gantenerumab), AMG 780, MEDI 3617, 네스바쿠맙 (Nesvacumab), 바누씨주맙 (Vanucizumab), 에비나쿠맙 (Evinacumab), 타발루맙 (Tabalumab), 라나루맙 (Lanalumab), 베리무맙 (Belimumab), 옴부르타맙 (omburtamab), 라불리주맙 (Ravulizumab), 에쿨리주맙 (Eculizumab), 모가물리주맙 (Mogamulizumab), 오텔릭시주맙 (Otelixizumab), 테플리주맙 (Teplizumab), 무로모납 (Muromonab), 테벤타프스프 (Tebentafusp), 블리나두모맙 (Blinatumoma), REGN1979, 이발리주맙 (Ibalizumab), 자놀리무맙 (Zanolimumab), 이톨리주맙 (Itolizumab), 에팔리주맙 (Efalizumab), 이네빌리주맙 (Inebilizumab), 타파시타맙 (Tafasitamab), 론카스툭시맙 테시린 (Loncastuximab tesirine), 오크렐리주맙 (Ocrelizumab), 우블리툭시맙 (Ublituximab), 오비누투주맙 (Obinutuzumab), 오파투무맙 (Ofatumumab), 리툭시맙 (Rituximab), 토시투모맙 (Tositumomab), 이브리투모맙 티욱세탄 (Ibritumomab tiuxetan), 에프라투주맙 (Epratuzumab), 이노투주맙 오조가마이신 (Inotuzumab ozogamicin), 목세투모맙 파수도톡스 (Moxetumomab pasudotox), 브렌툭시맙 베도틴 (Brentuximab vedotin), 바다스툭시맙 탈리닌 (Vadastuximab talirine), 젬투주맙 오조가마이신 (Gemtuzumab ozogamicin), 다라투무맙 (Daratumumab), 이사툭시맙 (Isatuximab), 알렘투주맙 (Alemtuxumab), 크리잔리주맙 (Crizanlizumab), 폴라투주맙 베도틴 (Polatuzumab vedotin), 갈릭시맙 (Galiximab), žƒ티네주맙 (Eptinezumab), 프레마네주맙 (Fremanezumab), 갈카네주맙 (Galcanezumab), 에레누맙 (Erenumab), 졸베툭시맙 (Zolbetuximab), 람팔리주맙 (Lampalizumab), 트레멜리무맙 (Tremelimumab), 잘리프렐리맙 (Zalifrelimab), 이플리무맙 (Ipilimumab), 로발피투주맙 테시린 (Rovalpituzumab tesirine), 세툭시맙 (Cetuximab), 데파툭시주맙 (Depatuxizumab), 잘루투무맙 (Zalutumumab), 네시투무맙 (Necitumumab), 파니투무맙 (Panitumumab), 에미시주맙 (Emicizumab), 니포칼리맙 (Nipocalimab), 로잔놀릭시주맙 (Rozanolixizumab), 부로스맙 (Burosumab), 팔레투주맙 (Farletuzumab), 미르베툭시맙 소랍탄신 (Mirvetuximab soravtansine), 디누툭시맙 (Dinutuximab), 낙시타맙 (Naxitamab), 오틸리맙 (Otilimab), 마르게툭시맙 (Margetuximab), 페르투주맙 (Pertuzumab), 트라스투주맙 (Trastuzumab), 트라수투주맙 데룩테칸 (Trastuzumab deruxtecan), 트라수투주맙 엠탄신 (Trastuzumab emtansine), 트라수투주맙 두오카르마진 (Trastuzumab duocarmazine), 파트리투맙 (Patritumab), 아니프롤루맙 (Anifrolumab), 에마팔루맙 (Emapalumab), 리겔리주맙 (Ligelizumab), 오말리주맙 (Omalizumab), 달로투주맙 (Dalotuzumab), 피기투무맙 (Figitumumab), 테프로투무맙 (Teprotumumab), 게보키주맙 (Gebokizumab), 카나키누맙(Canakinumab), 다클리주맙 (Daclizumab), 바실릭시맙(Basiliximab), 두필루맙 (Dupilumab), 메폴리주맙 (Mepolizumab), 레슬리주맙 (Reslizumab), 벤랄리주맙 (Benralizumab), 클라자키주맙 (Clazakizumab), 올로키주맙 (Olokizumab), 시루쿠맙 (Sirukumab), 실툭시맙 (Siltuximab), 사릴루맙 (Sarilumab), 사트랄리주맙 (Satralizumab), 토실리주맙 (Tocilizumab), REGN88, 세쿠키누맙 (Secukinumab), 우스테키누맙 (Ustekinumab), 브리아키누맙 (Briakinumab), 레브리키주맙 (Lebrikizumab), 트랄로키누맙 (Tralokinumab), 익세키주맙 (Ixekizumab), 비메키주맙 (Bimekizumab), 브로달루맙 (Brodalumab), 브라지쿠맙 (Brazikumab), 구셀쿠맙 (Guselkumab), 리산키주맙 (Risankizumab), 틸드라키주맙 (Tildrakizumab), 미리키주맙 (Mirikizumab), 네몰리주맙 (Nemolizumab), 스페솔리맙 (Spesolimab), 렐라틀리맙 (Relatlimab), 나르소플리맙 (Narsoplimab), 파시누맙 (Fasinumab), 타네주맙 (Tanezumab), 알리로쿠맙 (Alirocumab), 에볼로쿠맙 (Evolocumab), 보코시주맙 (Bococizumab), 람브롤리주맙 (Lambrolizumab), 발스틸리맙 (Balstilimab), 캄렐리주맙 (Camrelizumab), 쎄미플리맙 (Cemiplimab), 도스탈리맙 (Dostarlimab), 프롤골리맙 (Prolgolimab ), 신틸리맙 (Sintilimab), 스파르탈리주맙 (Spartalizumab), 티슬레리주맙 (Tislelizumab), 펨브롤리주맙 (Pembrolizumab), 니볼루맙 (Nivolumab), 아테졸리주맙 (Atezolizumab), 아벨루맙 (Avelumab), 엔바폴리맙 (Envafolimab), 둘발루맙 (Durvalumab), 빈트라푸스프 알파 (Bintrafusp alpha), 데노수맙 (Denosumab), 엘로투주맙 (Elotuzumab), 콘시주맙 (Concizumab), 말스타시맙 (Marstacimab), 인플릭시맙 (Infliximab), 아달리무맙 (Adalimumab), 골리무맙 (Golimumab), 세르톨리주맙 페골 (Certolizumab pegol), 오조랄리주맙 (Ozoralizumab), 브롤루시주맙 (Brolucizumab), 라니비주맙 (Ranibizumab), 베바시주맙 (Bevacizumab), 파리시맙 (Faricimab), 라무시루맙 (Ramucirumab), 및 카플라시주맙 (Caplacizumab)으로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 하는 약학적 조성물.
The method of claim 14, wherein the antibody is Utomilumab, Natalizumab, Etrolizumab, Vedolizumab, Bimagrumab, Bapineuzumab , Crenezumab, Solanezumab, Aducanumab, Gantenerumab, AMG 780, MEDI 3617, Nesvacumab, Vanucizumab, Evie Evinacumab, Tabalumab, Lanalumab, Belimumab, omburtamab, Ravulizumab, Eculizumab, Mogamulizumab (Mogamulizumab), Otelixizumab, Teplizumab, Muromonab, Tebentafusp, Blinatumoma, REGN1979, Ibalizumab, Zanolimumab, Itolizumab, Efalizumab, Inebilizumab, Tafasitamab, Loncastuximab tesirine, ocrelizumab (Ocrelizumab), Ublituximab, Obinutuzumab, Ofatumumab, Rituximab, Tositumomab, Ibritumomab Tiuxetan tiuxetan), Epratuzumab, Inotuzumab ozogamicin, Moxetumomab pasudotox, Brentuximab vedotin, vadastuximab talinin (Vadastuximab talirine), Gemtuzumab ozogamicin, Daratumumab, Isatuximab, Alemtuxumab, Crizanlizumab, Polatuzumab vedotin (Polatuzumab vedotin), Galiximab, Epitinezumab, Fremanezumab, Galcanezumab, Erenumab, Zolbetuximab, Lampalizumab (Lampalizumab), Tremelimumab, Zaliprelimab, Ipilimumab, Rovalpituzumab tesirine, Cetuximab, Depatuxizumab , Zalutumumab, Necitumumab, Panitumumab, Emicizumab, Nipocalimab, Rozanolixizumab, Burosumab, Farletuzumab, Mirvetuximab soravtansine, Dinutuximab, Naxitamab, Otilimab, Margetuximab, Pertuzumab (Pertuzumab), Trastuzumab, Trastuzumab deruxtecan, Trastuzumab emtansine, Trastuzumab duocarmazine, Patritumab, no Anifrolumab, Emapalumab, Ligelizumab, Omalizumab, Dalotuzumab, Figitumumab, Teprotumumab, Gevoki Gebokizumab, Canakinumab, Daclizumab, Basiliximab, Dupilumab, Mepolizumab, Reslizumab, Benralizumab ( Benralizumab, Clazakizumab, Olokizumab, Sirukumab, Siltuximab, Sarilumab, Satralizumab, Tocilizumab , REGN88, Secukinumab, Ustekinumab, Briakinumab, Lebrikizumab, Tralokinumab, Ixekizumab, Bimekizumab (Bimekizumab), Brodalumab, Brazikumab, Guselkumab, Risankizumab, Tildrakizumab, Mirikizumab, Nemolizumab ), Spesolimab, Relatlimab, Narsoplimab, Fasinumab, Tanezumab, Alirocumab, Evolocumab , Bococizumab, Lambrolizumab, Balstilimab, Camrelizumab, Cemiplimab, Dostarlimab, Prolgolimab, Sintilimab, Spartalizumab, Tislelizumab, Pembrolizumab, Nivolumab, Atezolizumab, Avelumab, Enva Envafolimab, Durvalumab, Bintrafusp alpha, Denosumab, Elotuzumab, Concizumab, Marstacimab ), Infliximab, Adalimumab, Golimumab, Certolizumab pegol, Ozoralizumab, Brolucizumab, Ranibizumab ( A pharmaceutical composition comprising at least one selected from the group consisting of Ranibizumab, Bevacizumab, Faricimab, Ramucirumab, and Caplacizumab.
제14항에 있어서, 상기 수용성 수용체 또는 수용성 수용체와 Fc 융합 단백질에 포함되는 수용성 수용체는 TNF-α 수용성 수용체, VEGF 수용성 수용체, CTLA-4, 인터루킨1 수용성 수용체, 및 LFA3 수용성 수용체로 이루어진 군에서 선택되는 것을 특징으로 하는 약학적 조성물.
The method of claim 14, wherein the soluble receptor or the soluble receptor included in the soluble receptor and Fc fusion protein is selected from the group consisting of TNF-α soluble receptor, VEGF soluble receptor, CTLA-4, interleukin 1 soluble receptor, and LFA3 soluble receptor. A pharmaceutical composition characterized in that:
제17항에 있어서, 상기 수용성 수용체와 Fc의 융합 단백질은 에타너셉트(Etanercept), 아플리버셉트 (Aflibercept), 아바타셉트 (Abatacept), 벨라다셉트(Belatacept), 릴로나셉트 (Rilonacept), 및 알레파셉트 (Alefacept)로 이루어진 군에서 선택되는 것을 특징으로 하는 약학적 조성물.
The method of claim 17, wherein the fusion protein of the soluble receptor and Fc is Etanercept, Aflibercept, Abatacept, Belatacept, Rilonacept, and Alefacecept. A pharmaceutical composition selected from the group consisting of (Alefacept).
제11항에 있어서, 완충제, 안정화제, 및 계면활성제로 이루어진 군에서 선택되는 어느 하나 이상을 추가로 포함하는 것을 특징으로 하는 약학적 조성물.
The pharmaceutical composition according to claim 11, further comprising at least one selected from the group consisting of a buffer, a stabilizer, and a surfactant.
제19항에 있어서,
상기 완충제는 말산염 (malate), 폼산염 (formate), 시트르산염 (citrate), 아세테이트 (acetate), 프로피오네이트 (propionate), 피리딘 (pyridine), 피페라진 (piperazine), 카코딜산 (cacodylate), 석신산 (succinate), 2-(N-모르폴리노)에탄설폰산 (2-(N-morpholino)ethanesulfonic acid, MES), 히스티딘 (histidine), 트리스 (Tris), 비스-트리스 (bis-Tris), 인산염 (phosphate), 에탄올아민 (ethanolamine), 탄산염 (carbonate), 2-에테인술폰산 (piperazine-N,N'-bis(2-ethanesulfonic acid), PIPES), 이미다졸 (imidazole), 비스-트리스 프로판 (BIS-TRIS propane), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (Hydroxyethyl piperazine Ethane Sulfonic acid), 피로인산염(pyrophosphate), 및 트리에탄올아민 (triethanolamine)으로 이루어진 군에서 선택되는 1종 이상의 완충제이고;
상기 안정화제는 탄수화물, 당류 또는 이들의 수화물, 당알콜류 또는 이들의 수화물, 및 아미노산으로 이루어진 군에서 선택되는 1종 이상이며;
상기 계면활성제는 폴리옥시에틸렌-소르비탄 지방산, 폴리에틸렌-폴리프로필렌 글리콜, 폴리옥시에틸렌-스테아레이트, 폴리옥시에틸렌 알킬 에테르, 폴리옥시에틸렌-폴리옥시프로필렌 공중합체, 및 나트륨 도데실 설페이트 (SDS)로 이루어진 군에서 선택되는 하나 이상의 비이온성 계면활성제인 것;
을 특징으로 하는 약학적 조성물.
According to clause 19,
The buffering agents include malate, formate, citrate, acetate, propionate, pyridine, piperazine, cacodylate, Succinate, 2-(N-morpholino)ethanesulfonic acid (MES), histidine, Tris, bis-Tris , phosphate, ethanolamine, carbonate, 2-ethanesulfonic acid (piperazine-N,N'-bis(2-ethanesulfonic acid), PIPES), imidazole, bis-tris propane (BIS-TRIS propane), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (Hydroxyethyl piperazine Ethane Sulfonic acid), pyrophosphate (pyrophosphate), and at least one buffer selected from the group consisting of triethanolamine;
The stabilizer is at least one selected from the group consisting of carbohydrates, saccharides or their hydrates, sugar alcohols or their hydrates, and amino acids;
The surfactant is polyoxyethylene-sorbitan fatty acid, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ether, polyoxyethylene-polyoxypropylene copolymer, and sodium dodecyl sulfate (SDS). One or more nonionic surfactants selected from the group consisting of;
A pharmaceutical composition characterized by:
제20항에 있어서,
상기 탄수화물, 당류 또는 당알콜류는 트레할로스 또는 그의 수화물, 수크로스, 사카린, 글리세롤, 에리스톨, 트레이톨, 자일리톨, 아라비톨, 리비톨, 만니톨, 소르비톨, 갈락티톨, 푸시톨, 아이디톨, 이노시톨, 볼레미톨, 이소말트, 말티톨, 폴리글리시톨, 사이클로덱스트린(cyclodextrin), 하이드로옥실프로필 사이클로덱스트린(Hydroxypropyl Beta-cyclodextrin), 및 글루코스로 이루어진 군에서 선택되는 1종 이상이고;
상기 아미노산은 글루타민, 글루탐산, 글라이신, 라이신, 라이실라이신, 류신, 메치오닌, 발린, 세린, 셀레노메치오닌, 시트룰린, 아르지닌, 아스파라진, 아스파트산, 오르니틴, 아이소류신, 타우린, 테아닌, 트레오닌, 트립토판, 타이로신, 페닐알라닌, 프롤린, 피롤라이신, 히스티딘, 및 알라닌으로 이루어진 군에서 선택되는 1종 이상인 것;
을 특징으로 하는 약학적 조성물.
According to clause 20,
The carbohydrates, saccharides, or sugar alcohols include trehalose or its hydrate, sucrose, saccharin, glycerol, erythritol, threitol, xylitol, arabitol, ribitol, mannitol, sorbitol, galactitol, fusitol, iditol, inositol, and bol. At least one selected from the group consisting of remitol, isomalt, maltitol, polyglycitol, cyclodextrin, hydroxypropyl cyclodextrin (Hydroxypropyl Beta-cyclodextrin), and glucose;
The amino acids include glutamine, glutamic acid, glycine, lysine, lysyllysine, leucine, methionine, valine, serine, selenomethionine, citrulline, arginine, asparagine, aspartic acid, ornithine, isoleucine, taurine, theanine, and threonine. , tryptophan, tyrosine, phenylalanine, proline, pyrrolysine, histidine, and alanine;
A pharmaceutical composition characterized by:
제11항에 있어서, 상기 약학적 조성물은 피하 투여용인 것을 특징으로 하는 약학적 조성물.The pharmaceutical composition according to claim 11, wherein the pharmaceutical composition is for subcutaneous administration.