GB2521355A

GB2521355A - Human targets I

Info

Publication number: GB2521355A
Application number: GB1322250.0A
Authority: GB
Inventors: Jasper Clube
Original assignee: Kymab Ltd
Current assignee: Kymab Ltd
Priority date: 2013-12-17
Filing date: 2013-12-17
Publication date: 2015-06-24
Also published as: GB201322250D0

Abstract

A method of treating or preventing a disease or condition in a human is disclosed, wherein the disease condition is mediated by a Target of Interest (TOI), wherein the TOI is present in humans as different polymorphic variants, the method comprising a) Administering to the human an anti-TOI ligand to target a TOI variant in the human and treat or prevent said disease or condition, wherein the TOI in said human is encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or wherein the TOI in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%; wherein b) Before step (a) said human has been or is genotyped as positive for said nucleotide sequence or phenotyped as positive for said TOI variant. The TOI may be Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9). Associated ligands, kits, and genotyping methods are also claimed.

Description

Intellectual Property Office Application No. GB1322250.O RTN4 Date:28 August 20t4 The following terms are registered trade marks and should be read as such wherever they occur in this document: Biacore Intellectual Property Office is an operating name of the Patent Office www.ipo.govuk

HUMAN TARGETS I

TECHNICAL FIELD

[0001] The technology described herein relates to ligands, e.g., antibodies for the treatment of disease,

BACKGROUND

[0002] It is recognized that individual humans differ in their sequence and recently several individuals have had their genomes sequenced, for instance James Watson and Craig Venter. Comparison of the genome sequence of individuals has revealed differences in their sequences in both coding and non-coding parts of the genome. Some of these variations in humans are significant and contribute to phenotypic differences between individuals, In extreme cases these will result in genetic disease, The OOO Genomes Project has the objective of cataloguing sequences in the human genome, involving sequencing the genomes of a very large sampling of individuals from diverse art-recognized human ethnic populations.

SUMMARY

[0003] Through the application of human genetic variation analysis and rationally-designed sequence selection the present invention provides for improved human patient diagnosis and therapy. Importantly, the invention enables tailored medicines that address individual human patient genotypes or phenotypes.

[0004] The inventor's analysis of large numbers of naturally-occurring genomic human TOl sequences reveals that there is significant variation across diverse human populations and provides for the ability for correlation between individual human patients and tailored medical and diagnostic approaches addressing the target. The technical applications of these findings, as per the present invention, thus contribute to better treatment, prophylaxis and diagnosis in humans and provides for patient benefit by enabling personalized medicines and therapies. This provides advantages of better prescribing, less wastage of medications and improved chances of drug efficacy and better diagnosis in patients.

[0005] Furthermore, the inventor surprisingly realised that some rarer natural forms, although present in humans at much lower frequencies than the common form, nevertheless are represented in multiple and ethnically-diverse human populations and usually with many human examples per represented ethnic population. Thus, the inventor realised that targeting such rarer forms would provide for effective treatment, prophylaxis or diagnosis across many human ethnic populations, thereby extending the utility of the present invention and better serving patients in those populations.

[0006] With this, the inventor realised that there is significant industrial and medical application for the invention in terms of guiding the choice of an anti-TOT ligand for administration to human patients for therapy and/or prophyl axis of TOt-mediated or associated diseases and conditions. In this way, the patient receives drugs and ligands that are tailored to their needs -as determined by the patient's genetic or phenotypic makeup.

Hand-in-hand with this, the invention provides for the genotyping and/or phenotyping of patients in connection with such treatment, thereby allowing a proper match of drug to patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment using drugs or ligands that are not matched to the patient (eg, poor efficacy and/or side-effects) and avoids pharmaceutical mis-prescription and waste.

[0007] To this end, the invention provides:- [0008] Tn a First Configuration [0009] A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOI), wherein the TOI is present in humans as different polymorphic variants, the method comprising a. Administering to the human an anti-TOT ligand to target a TOT variant in the human and treat or prevent said disease or condition, wherein the TOI in said human is encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or wherein the TOT in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%; wherein b. Before step (a) said human has been or is genotyped as positive for said nucleotide sequence or phenotyped as positive for said TOT variant.

[OOTO] In a Second Configuration [OOT1] A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Tnterest (TOI), wherein the TOI is present in humans as different polymorphic variants, the method comprising a. Administering to the human an anti-TOt ligand to target a TOt variant in the human and treat or prevent said disease or condition, wherein the TOI in said human is encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or wherein the TOL in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%; wherein b. before step (a) the ligand has been or is determined as capable of binding to said TOl variant.

[0012] In a Third Configuration [0013] A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOT), wherein the TOl is present in humans as different polymorphic variants, the method comprising a. Administering to the human an anti-TOl ligand to target a TOl variant in the human and treat or prevent said disease or condition, wherein the TOT in said human is a variant encoded by a nucleotide sequence having a cumulative human allele frequency of more than 50% and/or having a total human genotype frequency of more than 50%; wherein b, Before step (a) said human has been or is genotyped as negative for a variant nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; or phenotyped as negative for a TOT variant encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

[0014] In a Fourth Configuration [0015] An anti-human TOT ligand for use in a method of treating and/or preventing a TOt-mediated disease or condition in a human, wherein the TOI is present in humans as different polymorphic variants and wherein the genome of said human comprises a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, the method comprising administering the ligand to the human.

[0016] Iii a Fifth Configuration [0017] A ligand that binds a human TOl comprising an amino acid sequence encoded by a TOt nucleotide sequence having a cumulative human allele frequency of less than 50% andlor a total human genotype frequency of less than 50%, for use in a method comprising the step of using the ligand to target said TOI in a human to treat and/or prevent a disease or condition mediated by TOT, the method comprising administering the ligand to the human.

[0018] In a Sixth Configuration [0019] A pharmaceutical composition or kit for treating or preventing a condition or disease mediated by a TOT.

[0020] In a Seventh Configuration [0021] A method of producing an anti-human TOL antibody binding site, the method comprising obtaining a plurality of anti-TOl antibody binding sites, screening the antibody binding sites for binding to a TOT comprising an amino acid sequence encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, or to a peptide thereof that comprises an amino acid variation from the corresponding sequence encoded by the TOL-encoding nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency, and isolating an antibody binding site that binds in the screening step.

[0022] In a Eighth Configuration [0023] A method of producing an anti-human TOI antibody, the method comprising immuni sing a non-human vertebrate (eg, a mouse or a rat) with a TOt comprising an amino acid sequence encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, or to a peptide thereof that comprises an amino acid variation from the corresponding sequence encoded by the 101-encoding nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency, and isolating an antibody that binds a TOT comprising an amino acid sequence encoded by a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, and optionally producing a TOI-binding fragment or derivative of the isolated antibody.

[0024] In a Ninth Configuration [0025] A kit for TOT genotyping a human, wherein the kit comprises a nucleic acid comprising a nucleotide sequence that specifically hybridises to a 101 nucleotide sequence selected having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or an RNA transcript thereof and/or the nucleic acid comprises a nucleotide sequence that comprises at least 10 contiguous nucleotides of a 101 nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or is an antisense sequence thereof [0026] In a Tenth Configuration [0027] Use of an anti-TOl ligand that binds a human 101 comprising an amino acid sequence encoded by a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, in the manufacture of a medicament for treating and/or preventing a TOT-mediated disease or condition in a human whose genome comprises a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

[0028] In a Eleventh Configuration [0029] Use of an anti-TOT ligand that binds a human TOI comprising an amino acid sequence encoded by a TOt nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, in the manufacture of a medicament for targeting said TOl in a human to treat and/or prevent a disease or condition mediated by TOL.

[0030] In a Twelfth Configuration [0031] A method of targeting a TOI for treating and/or preventing a TOT-mediated disease or condition in a human, the method comprising administering an anti-TOl ligand to a human comprising a TOl nucleotide sequence selected having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, whereby a TOT encoded by said nucleotide sequence is targeted.

[0032] In a Thirteenth Configuration [0033] A method of TOt genotyping a nucleic acid sample of a human, the method comprising identifying in the sample the presence of a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

[0034] In a Fourteenth Configuration [0035] A method of TOT typing a protein sample of a human, the method comprising identifying in the sample the presence of a TOl amino acid sequence encoded by a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

[0036] Tn an example, the TOT is a human TOT selected from the group consisting of PCSK9, VEGF-A and lL6 receptor.

BRTEF DESCRTPTTON OF THE DRAWINGS

[0037] This patent or application file contains at least one drawing executed in color.

Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0038] Figure 1 shows in silica modeling of PCSK9 surface variant residues.

[0039] Figure 2 depicts the cumulative allele frequency distribution across the 1000 Genomes Project databse of human VH3-23 alleles comprising SNP rs56069819 (such alleles denonted "C" and the most frequent allele (which does not comprise this SNP) denoted "A").

The figure shows that VH3-23 alleles comprising SNP rs56069819 are present at a cumulative frequency of 11% across all human ethnic populations taken as a whole, whereas in certain specific human ethnic sub-populations (ASW, LWK, YRI, CPU and GBR) such alleles are present at an above-average cumulative frequency. Indicated in the figure are those human PCSK9 variant forms (marked "Variants") that are found in the various sub-populations with above-average occurence of human Vt-l3-23 alleles comprising SNP rs560698 19.

[0040] Figure 3 depicts frameworks and CDRs encoded by VH323*04 as obtained from the IMGT database (available on the World Wide Web at www.tMGT.org).

[0041] Figure 4 depicts sequences of VH323*04, The portion of VH323*04 comprising the FWI residue change ofrs56069819 (SEQ ID NO: 38). The portion of the nucleic acid sequence encoding rs56069819 is depicted (SEQ ID NO: 39). The FWI encoded by V1I323*04 is depicted (SEQ ID NO: 40).

DETAILED DESCRIPTION

[0042] The skilled person will know that SNPs or other changes that translate into amino acid variation can cause variability in conformation or activity of human targets to be addressed. This has spawned great interest in personalized medicine where genotyping and knowledge of protein and nucleotide variability is used to tailor medicines and diagnosis of patients more effectively. The present invention provides for tailored pharmaceuticals and testing that specifically addresses rarer variant forms of a human target of interest (Tot).

[0043] The present invention harnesses the power of human genetic variation analysis and rationally-designed sequence selection, The technical applications of these approaches, as per the present invention, contribute to better treatment, prophylaxis and diagnosis in humans and provides for patient benefit by providing choice and enabling personalized medicines and therapies. This provides advantages of better prescribing, less wastage of medications and improved chances of drug efficacy and better diagnosis in patients.

As sources of genomic sequence variation data, the skilled person will be aware of the available databases and resources (including updates thereof) provided by the following:- 1. HapMap (The International HapMap Consortium. 2003; available on the \Vorld Wide Web at http://hapmap.ncbi.nlm.nih.gov/index.html en). The HapMap Project is an international project that aims to compare the genetic sequences of different individuals to identify chromosomal regions containing shared genetic variants. The HapMap www site provides tools to identify chromosomal regions and the variant therein, with options to drill down to population level frequency data.

2. 1000 Genomes Project (The 1000 Genomes Project Consortium 2010; available on the World Wide Web at http://www.l000genomes.org!). This resource provides complete genomic sequence for at least 2500 unidentified individuals from one of 25 distinct population groups.

3. Japanese SNP Database( FiHaga et al, 2002; available on the World Wide Web at http://snp.ims.u-tokyo.ac.jp/index.html). Based on a study identifying 190,562 human genetic variants.

[0044] The present invention involves the identification and cataloguing of naturally-occurring human genomic target sequence variants, including those found to be relatively low-frequency or rare variants that segregate with specific human ethnic populations and in many individual humans.

[0045] An aspect of the invention is based on rational design of sequence selection addressing the desirability to tailor medicaments and diagnostics to rarer, but yet still significant groups of human individuals that suffer from, or have the potential to suffer from (ie, who are at risk of), a disease or condition mediated or associated with the target of interest, In devising this rational design of the present aspect of the invention, the inventor included considerations of the spread of prevalence of naturally-occurring target variant sequences across multiple, diverse human ethnic populations, as well as the importance of addressing such populations where many individuals are likely to display a genotype and/or phenotype of one or more of the variants being analysed. As part of this design, the inventor saw the importance of adopting the art-recognised classifications of human ethnic populations, and in this respect the inventor based the analysis and design on the recognised human ethnic populations adopted by the 1000 Genomes Project, since this is a resource that is, and will continue to be, widely adopted by the scientific and medical community.

[0046] Thus, in this aspect of the invention, the inventor designed the following variant sequence selection criteria, these being criteria that the inventor realised would provide for useful medical drugs and diagnostics to tailored need in the human population.

[0047] Selection Criteria [0048] Three or four of the following:-a * Naturally-occurring human target variant sequences having a cumulative human allele frequency of 35% or less; * Naturally-occurring human target variant sequences having a total human genotype frequency of 40% or less; * Naturally-occurring human target variant sequences found in many different human ethnic populations (using the standard categorisation of the 1000 Genomes Project; see Table 3 below); and * Naturally-occurring human target variant sequences found in many individuals distributed across such many different ethnic populations.

[0049] The inventor's selection included, as a consideration, selection for nucleotide variation that produced amino acid variation in corresponding 101 forms (ie, non-synonymous variations), as opposed to silent variations that do not alter amino acid residues in the target protein.

[0050] In an embodiment, the cumulative human allele frequency is 30, 25, 20, 15, 10 or 5% or less, eg, in the range from ito 20% or Ito 15% or ito 10%.

[0051] In an embodiment, the total human genotype frequency is 35, 30, 25, 20, 15, 10 or 5% or less, eg, in the range from ito 25%, 1 to 20%, ito 15%, Ito about 15%, 1 to 10%, 1 to about iO% or ito 5% or Ito about 5%.

[0052] In an embodiment, the naturally-occurring human target variant sequences are found in at least 9, 10, ii, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different human ethnic populations (using the standard categorisation of the 1000 Genomes Project).

[0053] In an embodiment, the naturally-occurring human target variant sequences are found in at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 iO, 115, 120, 30, 140 or 150 individuals distributed across such many different ethnic populations.

[0054] lii an example, the following criteria are applied:- * Naturally-occurring human target variant sequences having a cumulative human allele frequency of 15% or less; * Naturally-occurring human target variant sequences having a total human genotype frequency of 20% or less; * Naturally-occurring human target variant sequences found in at least 5 different human ethnic populations (using the standard categorisation of the 1000 Genomes Proj ect); and Naturally-occurring human target variant sequences found in many individuals distributed across such many different ethnic populations.

100551 In an example, the criteria are applied with reference to one or more human genomic sequence databases as described herein. For example, the criteria are those as applied to the 1000 Genomes database.

[0056] For example in any aspect example, embodiment or configuration of the invention, the 1000 Genomes database release 13. For example, the 1000 Genomes database in its most recent version as at 1 October 2013.

[0057] Optionally, further sequence analysis and 3D in si//co modelling (eg, see Figure 1) can also be used as an additional selection criterion: variants whose variant amino acid residues (versus the most common form of human TOT) are surface-exposed on the target are desirable for selection, since the inventor saw these as contributing to determining the topography of the target and potentially contributing to how and where ligand binding on the target occurs.

[0058] The following bioinformatics protocol is envisaged to indentify human sequences for use in the present invention: (a) Identify a genomic region containing a target sequence of interest (target genomic region') and calculate the genomic coordinates, using coordinates that match the sequence assembly build used by either the 1000 Genomes Project or International I-lapMap project (or another selected human gene database of choice).

b) Identify genomic variants mapped to the genomic region previously identified in (a).

Retrieve allele frequencies for variants for each super population and preferably sub-population where such data is available. The VWC tools for the 1000 Genomes Project can be used for this step.

(c) Filter list of genomic variants from target genomic region to contain only variants classed as either non-synonymous' single nucleotide polymorphisms (SNP5) or genomic insertions or delections' (indels). Filter further to include those that are present in exonic sequences only. "Non-synonymous" refers to nucleotide variation that produces amino acid variation (ie, excluding silent mutations).

(d) Correlate population frequency data for each of the identified variants for each of the super populations (for example European Ancestry', East Asian ancestry', West African ancestry', Americas', and South Asian ancestry') to identify those variants that segregate with less than two super-populations. Further correlate all identified variants with each of the sub-populations (for example, European ancestry' super-population might be subdivided into groups such as CEU -Utah residents with Northern or Western European ancestry', TSI Toscani in Italia' and British from England and Scotland') and produce a second score for rarity of variants within a super-population.

(e) Collect one or more sequences that show segregation to specific sub-populations for use in the present invention, eg, according to selection criteria as described herein.

[0059] 1-tuinan Populations [0060] Optionally the ethnic populations are selected from those identified in the 1000 Genomes Project database. In this respect, see Table 3 which provides details of the ethnic populations on which the 1000 Genomes Project database is based.

[0061] N A Rosenberg eta! (Science 20 December 2002: vol. 298 no, 5602 2342-2343) studied the genetic structure of human populations of differing geographical ancestry. In total, 52 populations were sampled, these being populations with: [0062] African ancestry [0063] (Mbuti Pygmies, Biaka Pygmies, San peoples, and speakers of Niger-Kordofanian languages (Bantu, Yoruba or Mandenka populations), [0064] Eurasian ancestry [0065] (European ancestry (Orcadian, Adygel, Basque, French, Russians, Italians, Sardinian, Tuscan), [0066] Middle Eastern ancestry (Mozabite, Bedouin, Druze, Palestinians), [0067] Central/South Asian ancestry (Balochl, Brahul, Makrani, Sindhi, Pathan, Burusho, Hazara, Uygur, Kalash)), [0068] Thsi As/at, ancestry [0069] (Han, Dal, Daur, Hezhen, Lahu, Miao, Oroqen, She, Tujia, Tu, Xibo, Yi, Mongola, Naxi, Cambodian, Japanese, Yakut), Oceanic ancestry (Melanesian. Papuan); or [0070] Americas ancestry [0071] (Karitiana, Surui, Colombian, Maya, Pima).

[0072] The International HapMap Project, Nature, 2003 Dec 18;426(6968):789-96, discloses that goal of the HapMap Project: to determine the common patterns of DNA sequence variation in the human genome by determining the genotypes of one million or more sequence variants, their frequencies and the degree of association between them in DNA samples from populations with ancestry from parts of Africa, Asia and Europe. The relevant human populations of differing geographical ancestry include Yoruba, Japanese, Chinese, Northern European and Western European populations. More specifically:- [0073] Utah population with Northern or Western European ancestry (samples collected in 1980 by the Centre d'Etude du Polymorphisme Humain (CEPH); population with ancestry of Yoruba people from Ibadan, Nigeria; population with Japanese ancestry; and population with ancestry of Han Chinese from China.

[0074] The authors, citing earlier publications, suggest that ancestral geography is a reasonable basis for sampling human populations.

[0075] A suitable sample of human populations used in the present invention is as follows:- (a) European ancestty (b) Northern European ancestry; Western European ancestry; Toscani ancestry; British ancestry, Finnish ancestry or Iberian ancestry.

(c) More specifically, population of Utah residents with Northern and/or Western European ancestry; Toscani population in Italia; British population in England and/or Scotland; Finnish population in Finland; or Iberian population in Spain.

(a) East Asian ancestry (b) Japanese ancestry; Chinese ancestry or Vietnamese ancestry.

(c) More specifically, Japanese population in Toyko, Japan; Han Chinese population in Beijing, China; Chinese Dai population in Xishuangbanna; Kinh population in Ho Chi Minh City, Vietnam; or Chinese population in Denver, Colorado, USA.

(a) West African ancestry (b) Yoruba ancestry; Luhya ancestry; Gambian ancestry; or Malawian ancestry.

(c) More specifically, Yoruba population in Ibadan, Nigeria; Luhya population in Webuye, Kenya; Gambian population in Western Division, The Gambia; or Malawian popu'ation in Blantyre, Malawi.

(a) Population ?f ihe Americas b) Native American ancestry; Afro-Caribbean ancestry; Mexican ancestry; Puerto Rican ancestry; Columbian ancestry; or Peruvian ancestry.

(c) More specifically, population of African Ancestry in Southwest US; population of African American in Jackson, MS; population of African Caribbean in Barbados; population of Mexican Ancestry in Los Angeles, CA; population of Puerto Rican in Puerto Rico; population of Colombian in Medellin, Colombia; or population of Peruvian in Lima, Peru.

(a) South Asia,; ancestry (b) Ahom ancestry; Kayadtha ancestry; Reddy ancestry; Maratha or Punjabi ancestry.

(c) More specifically, Ahom population in the State of Assam, India. Kayadtha population in Calcutta, India Reddy population in Hyderabad, India; Maratha population in Bombay, India or Punjabi population in Lahore Pakistan.

100761 In any configuration of the invention, in one embodiment, each human population is selected from a population matted "(a)" above.

[0077] In any configuration of the invention, in another embodiment, each human population is selected from a population matted "(b)" above.

[0078] In any configuration of the invention, in another embodiment, each human population is selected from a population marked "(c)" above.

100791 In one embodiment the ethnic populations are selected from the group consisting of an ethnic population with European ancestry, an ethnic population with East Asian, an ethnic population with West African ancestry, an ethnic population with Americas ancestry and an ethnic population with South Asian ancestiy.

100801 In one embodiment the ethnic populations are selected from the group consisting of an ethnic population with Northern European ancesiry; or an ethnic population with Western European ancestry; or an ethnic population with Toscani ancestry; or an ethnic population with British ancesiry; or an ethnic population with Icelandic ancestry; or an ethnic population with Finnish ancestry; or an ethnic population with iberian ancestry; or an ethnic population with Japanese ancestry; or an ethnic population with Chinese ancestry; or an ethnic population Vietnamese ancestry; or an ethnic population with Yoniba ancestry; or an ethnic population with Luhya ancestry; or an ethnic population with Gambian ancestry; or an ethnic population with Malawian ancestry; or an ethnic population with Native American ancestry; or an ethnic population with Afro-Caribbean ancestry; or an ethnic population with Mexican ancestry; or an ethnic population with Puerto Rican ancestry; or an ethnic population with Columbian ancestry; or an ethnic population with Peruvian ancestry; or an ethnic population with Ahom ancestry; or an ethnic population with Kayadtha ancestry; or an ethnic population with Reddy ancestry; or an ethnic population with Maratha. or an ethnic population with Punjabi ancestry.

[0081] Anti-Target Ligands [0082] The invention provides useful and-target ligands for addressing humans suffering from or likely to suffer from a disease or condition mediated or associated with the TO!. For example, the ligand specifically binds to the TO! variant as per the invention. The ligand may inhibit or antagonise the activity of the target, eg, the ligand neutralises the target. The skilled person will be familiar with neutralising ligands in general, such as antibodies or antibody fragments, and can readily test suitable ligands for specific binding and/or neutralisation of a target En vitro or in an En vivo assay.

[0083] An antibody "fragment" comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include dAb, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0084] In an embodiment, the ligand of the invention is or comprises an antibody or antibody fragment, for example an antibody or fragment comprising human variable regions (and optionally also human constant regions). Anti-TOT or TOT-binding or targeting antibodies and fragments can be prepared according to any known method, eg, using transgenic mice (eg, the Kymousexl or Velocimouse'TM, or Omnimouse'TM, Xenomouse'1, HuMab MouseThi or MeMo Mousetmi), rats (eg, the Omniratm5, camelids, sharks, rabbits, chickens or other non-human animals immunised with the TOT followed optionally by humanisation of the constant regions and/or variable regions to produce human or humanised antibodies. In an example, display technologies can be used, such as yeast, phage or ribosome display, as will be apparent to the skilled person. Standard affinity maturation, eg, using a display technology, can be performed in a further step after isolation of an antibody lead from a transgenic animal, phage display library or other library. Representative examples of suitable technologies are described in US2O T 200938 T 8 (Amgen, Tnc), which is incorporated herein by reference, eg, the methods set out in paragraphs [0309] to [0346].

Although this is with reference to PCSK9, the antibody-generating methods can be applied to other TOls as per the broadest scopes of the present invention.

[0085] Generally, a VELOC1t'v1TvIIJNE' or other mouse or rat can be challenged with the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies, The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimaeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.

[0086] Initially, high affinity chimaeric antibodies are isolated having a human variable region and a mouse constant region. As described below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgGI or IgG4 (for example, SEQ ID NO: 75, 752,753 in US2O] 1/0065902, which sequences are incorporated herein by reference for use in the ligands of the present invention). While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.

[0087] Tn an example, the ligand of the invention is or comprises a nucleic acid, eg, RNA, eg, siRNA that hybridises under stringent condition to the TOT variant sequence, eg, hybridises a nucleotide sequence comprising one or more nucleotides that are variant (versus the most common TOt sequence, eg, with reference to the 1000 Genomes Project database).

[0088] Target binding ability, specificity and affinity (lcd, K0ff and!or K0) can be determined by any routine method in the art, eg, by surface plasmon resonance (SPR). The term "Kd", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0089] Tn one embodiment, the surface plasmon resonance (SPR) is carried out at 25°C.

In another embodiment, the SPR is carried out at 37°C.

[0090] In one embodiment, the SPR is carried out at physiological pH, such as about p1-17 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (also referred to as FIBS-EP)).

[0091] Tn one embodiment, the SPR is carried out at a physiological salt level, eg, 150mM NaC1, [0092] In one embodiment, the SPR is carried out at a detergent level of no greater than 0.05% by volume, eg, in the presence of P20 (polysorbate 20; eg, Tween-20Th) at 0.05% and EDTA at 3mM, [0093] In one example, the SPR is carried out at 25°C or 37°C in abufferat pH7.6, 150mM NaC1, 0.05% detergent (eg, P20) and 3mM EDTA. The buffer can contain 10mM Hepes. In one example, the SPR is carried out at 25°C or 37°C in HBS-EP. HBS-EP is available from Teknova Inc (California; catalogue number H8022).

[0094] In an example, the affinity of the ligand (eg, antibody) is determined using SPR by 1. Coupling anti-mouse (or other relevant human, rat or non-human vertebrate antibody constant region species-matched) IgG (eg, BiacoreThi BR-1008-38) to a biosensor chip (eg, GLM chip) such as by primary amine coupling; 2. Exposing the anti-mouse IgG (or other matched species antibody) to a test lgG antibody to capture test antibody on the chip; 3. Passing the test antigen over the chip's capture surface at 1O24nM, 256nM, 64nM, t6nM, 4nM with a OnM (i.e. buffer alone); and 4. And determining the affinity of binding of test antibody to test antigen using surface plasmon resonance, eg, under an SPR condition discussed above (eg, at 25°C in physiological buffer). SPR can be carried out using any standard SPR apparatus, such as by Biacoretmt or using the ProteOn XPR36ThT (Bio-Rad®).

[0095] Regeneration of the capture surface can be carried out with 10mM glycine at pHi.7. This removes the captured antibody and aflows the surface to be used for another interaction. The binding data can be fitted to t: t model inherent using standard techniques, eg, using a model inherent to the ProteOn XPR3&M analysis software.

[0096] In an example, the ligand of the invention is contained in a medical container, eg, a vial, syringe, IV container or an injection device (eg, an intraocular or intravitreal injection device). In an example, the ligand is in vitro, eg, in a sterile container. In an example, the invention provides a kit comprising the ligand of the invention, packaging and instructions for use in treating or preventing or diagnosing in a human a disease or condition mediated by the 101. h an example, the instructions indicate that the human should be genotyped for a TOT variant sequence of the invention before administering the ligand to the human. In an example, the instructions indicate that the human should be phenotyped for a TOt variant of the invention before administering the ligand to the human. In an example, the human is of Chinese (eg, Han) ethnicity and the instructions are in Chinese (eg, Mandarin), In an example, the instructions comprise directions to administer alirocumab or evolocumab to said human.

[0097] The invention relates to the concepts set out in the following clauses.

[0098] Clause t A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOt), wherein the TOt is present in humans as different polymorphic variants, the method comprising a. Administering to the human an anti-TOT ligand to target a TOT variant in the human and treat or prevent (eg, by at least 40, 50, 60, 70, 80, 90 or 5%) said disease or condition, wherein the TOt in said human is encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or wherein the TOT in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%; wherein Before step (a) said human has been or is genotyped as positive for said nucleotide sequence or phenotyped as positive for said TOT variant, or the method comprises before step (a) genotyping the human as positive for said nucleotide sequence or phenotyping the human as positive for said TOl variant.

[0099] In any aspect, configuration, example, embodiment, clause or concept herein, frequencies may be determined using bioinformatics.

[001001 In any aspect, configuration, example, embodiment, clause or concept herein, frequencies may be determined by reference to a database comprising at least 1000 or 2000 human sequences.

[001011 In any aspect, configuration, example, embodiment, clause or concept herein "heterozygous human genotype frequency" means the cumulative frequency of all genotypes in the sample or database or in humans having one occurrence of the rare variant allele and one occurrence of another allele (heterozygous state), eg, genotype in T ODD Genomes database.

[001021 In any aspect, configuration, example, embodiment, clause or concept herein "homozygous human genotype frequency means the cumulative frequency of two occurrences of the variant allele (homozygous state), eg, genotype in TODD Genomes Project database.

[001031 In any aspect, configuration, example, embodiment, clause or concept herein "total human genotype frequency" means the total of heterozygous plus homozygous human genotype frequencies.

[001041 In any aspect, configuration, example, embodiment, clause or concept herein "cumulative human allele frequency" refers to the total of all occurrences of the variant allele in the sample or database or in humans, eg, in the TODD Genomes Project database.

[001051 Clause 2: The method of clause I, wherein before step (a) the ligand has been or is determined as being capable of binding to said TOl variant, eg, with an affinity (Kd) disclosed below.

[001061 In an example, the ligand is (or has been determined as) a neutraliser of the TOT.

In an example, determination is carried out in a human (eg, in a clinical trial). In an example, determination is carried out in a non-human, eg, in a mouse, rat, rabbit, pig, dog, sheep or non-human primate (eg, Cynomolgous monkey, rhesus monkey or baboon).

1001071 Clause 3: A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TO!), wherein the TO! is present in humans as different polymorphic variants, the method comprising a. Administering to the human an anti-TO! ligand to target a TO! variant in the human and treat or prevent (eg, by at least 40, 50, 60, 70, 80, 90 or 5%) said disease or condition, wherein the TOt in said human is encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or wherein the TOt in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%; wherein b. Before step (a) the ligand has been or is determined as capable of binding to said TOI variant, eg, with an affinity (Kd) disclosed below, [00!08I In an example, the ligand is (or has been determined as) a neutraliser of the TOT, In an example, determination is carried out in a human (eg, in a clinical trial), In an example, determination is carried out in a non-human, eg, in a mouse, rat, rabbit, pig, dog, sheep or non-human primate (eg, Cynomolgous monkey, rhesus monkey or baboon).

[001091 Clause 4: The method of clause 3, wherein the genome of said human comprises a nucleotide sequence encoding said TOt variant; and before step (a) said nucleotide sequence has been or is determined as having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

[001101 The TO! variant is not the most frequent.

Fool Ill Clause 5: The method of clause 3 or 4, wherein said human has been or is genotyped as positive for said variant nucleotide sequence before step (a), or the method comprises genotyping the human as positive for said variant nucleotide sequence before step (a).

[00!121 Clause 6: The method of any preceding clause, wherein the human has been or is phenotyped as positive for said TOl variant before step (a), or the method comprises phenotyping the human as positive for said variant nucleotide sequence before step (a).

[001131 Clause 7: The method of any preceding clause, wherein said frequency is less than TO or!5% (eg, from! to!0%).

FOOtt4I In an embodiment, the cumulative human allele frequency is 30, 25, 20, 15, 10 or 5% or less, eg, in the range from! to 20% or t to t5% or ito 10%.

Fool 151 In an embodiment, the total human genotype frequency is 35, 30, 25, 20, 15, 10 or 5% or less, eg, in the range from ito 25%, ito 20%, ito 15%, Ito about 15%, ito 10%, 1 to about 1O?/ or ito 5% or I to about 5%.

[001161 Clause 8: The method of any preceding clause, wherein the ligand is capable of binding to two or more different TO! variants, each being encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% (eg, from Ito th%) and/or having a total human genotype frequency of less than 50% (eg, from ito 20%).

Fool 171 In an embodiment, the cumulative human allele frequency of each TOt variant is 30, 25, 20, 15, 10 or 5% or less, eg, in the range from Ito 20?/o or Ito 15% or Ito 10%, [001181 in an embodiment, the total human genotype frequency of each TO! variant is 35, 30, 25, 20, 15, 10 or 5% or less, eg, in the range from ito 25%, Ito 20%, ito i5%, ito about 15%, Ito 10%, 1 to about 10% or I to 5% or Ito about 5%, [001191 Clause 9: A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TO!), wherein the TO! is present in humans as different polymorphic variants, the method comprising a. Administering to the human an anti-TO! ligand to target a TO! variant in the human and treat or prevent (eg, by at least 40, 50, 60, 70, 80, 90 or 95%) said disease or condition, wherein the TOl in said human is a variant encoded by a nucleotide sequence having a cumulative human allele frequency of more than 50% (eg, the highest frequency) and/or having a total human genotype frequency of more than 50% (eg, the highest frequency); wherein b. Before step (a) said human has been or is genotyped as negative for a variant nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; or phenotyped as negative for a TOl variant encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; or Before step (a) said the method comprises genotyping the human as negative for a variant nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; or phenotyping the human as negative for a TO! variant encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

[001201 In an embodiment, in (a) the cumulative human allele frequency is 55, 60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%).

[001211 In an embodiment, in (a) the total human genotype frequency is 55, 60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%).

Fool 221 In an embodiment, in (b) the cumulative human allele frequency is 30, 25, 20, 15, or 5% or less, eg, in the range from ito 20% or 1 to 15% or 1 to 10%.

[001231 lii an embodiment, in (b) the total human genotype frequency is 35, 30, 25, 20, 15, or 5% or less, eg, in the range from to 25%, Ito 20%, to 15%, Ito about 15%, Ito 10%, Ito about 10% or to 5% or Ito about 5%, [001241 Clause 10: The method of clause 9, wherein before step (a), the human has been or is phenotyped as positive for the most frequent TOI variant or genotyped for the nucleotide sequence thereof [001251 In an embodiment, before step (a) the human has been oris genotyped as positive for TOT variant nucleotide sequence having a cumulative human allele frequency of 55, 60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%) or phenotyped for the TOt variant thereof [001261 Tn an embodiment, before step (a) the human has been or is genotyped as positive for TOT variant nucleotide sequence having a total human genotype frequency of 55, 60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or T00%(eg, in the range from 51 to 80%) or phenotyped for the TOt variant thereof [001271 Clause 11: The method of clause 9 or 10, wherein before step (a) the ligand has been or is determined as being capable of binding to the most frequent TOT variant.

[001281 Clause 12: The method of clause 9, 10 or II, wherein before step (a) the ligand has been or is determined as being substantially incapable of neutralising or inhibiting said TOI variant recited in step (b).

[001291 By "substantially incapable or neutralising or inhibiting" is meant: Neutralisation or inhibition less than 50, 25, 10, 5 or 0.5% inhibition or neutralisation of the most frequent TOI variant.

[001301 Clause 13: The method of any one of clauses 9 to 12, wherein the ligand is capable of binding to the most frequent TOI variant.

1001311 Clause 14: The method of any one of clauses 9 to 13, wherein the ligand is capable of binding to two or more different TOT variants, each being encoded by a nucleotide sequence having a cumulative human allele frequency of more than 50%.

1001321 In an embodiment, each TO! variant is encoded by a nucleotide sequence having a cumulative human allele frequency of 55, 60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%).

1001331 Tn an embodiment, each TOT variant is encoded by a nucleotide sequence having a total human genotype frequency of 55, 60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80?/), 1001341 Clause 15: The method of any preceding clause, wherein said variant nucleotide sequence recited in step (a) has been or is determined as being present in at least 2 different human ethnic populations, eg, at least 2, 3, 4, 5, 6, 7, 8 or 9 different human ethnic populations in Table 3.

1001351 Clause 16: The method of any preceding clause, wherein said human frequency is the frequency in a database of naturally-occurring sequences sampled from at least 15, 20 or different human ethnic populations and comprising at least 1000 sequences. In an embodiment, the database is the 1000 Genomes Project database as described herein.

1001361 Clause 17: An anti-human TOT ligand for use in a method of treating and/or preventing a TOT-mediated disease or condition in a human, wherein the TOT is present in humans as different polymorphic variants and wherein the genome of said human comprises a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, the method comprising administering the ligand to the human.

1001371 In the alternative, clause 17 provides an anti-human TOI ligand for use in a method according to any one of clauses 1 to 16, the method comprising administering the ligand to the human.

1001381 In an embodiment, the cumulative human allele frequency is 30, 25, 20, 15, 10 or 5% or less, eg, in the range from Ito 20% or Ito 15% or ito 10%.

1001391 Tn an embodiment, the total human genotype frequency is 35, 30, 25, 20, T5, TO or 5% or less, eg, in the range from Ito 25%, T to 20%, Ito 15%, Ito about 15%, T to 10%, 1 to about 0% or to 5% or Ito about 5%.

1001401 Clause 18: The ligand of clause 17, wherein the ligand has been or is determined as capable of binding the human TOT encoded by said nucleotide sequence.

1001411 In the alternative, clause S provides a ligand that binds a human TOL comprising an amino acid sequence encoded by a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% andlor a total human genotype frequency of less than 50%, for use in a method comprising the step of using the ligand to target said TO! in a human to treat and/or prevent a disease or condition mediated by TOT, the method comprising administering the ligand to the human.

[00t421 Clause 19: A ligand that binds a human TOT comprising an amino acid sequence encoded by a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, for use in a method according to any one of clauses 1 to T 6, the method comprising administering the ligand to the human.

[001431 Clause 20: The ligand of any one of clauses L/ to 19, wherein the human has been or is genotyped as positive for said TOI nucleotide sequence having a cumulative human allele frequency of less than 50%.

[001441 The ligand of any one of clauses 17 to 19, wherein the human has been or is genotyped as positive for said TOl nucleotide sequence having a total human genotype frequency of less than 50%, [001451 Clause 21: The ligand of any one of clauses 17 to 20, wherein the human has been or is phenotyped as positive for a TOT encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

[001461 Clause 22: The ligand of any one of clauses 17 to 2T, wherein the human has been or is genotyped as heterozygous for a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; optionally wherein the human has been or is genotyped as comprising a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and a TOT nucleotide sequence having a cumulative human allele frequency of more than 50% (eg, having the highest cumulative human allele frequency) and/or having a total human genotype frequency of more than 50% (eg, having the highest total human genotype frequency).

[001471 Clause 23: The ligand of any one of clauses 17 to 22, wherein the genome of the human has been or is genotyped as homozygous for a TOt nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

1001481 Clause 24: The ligand of any one of clauses L/ to 23, wherein the ligand comprises an antibody binding site that binds a human TOT comprising an amino acid sequence encoded by a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; and optionally has been or is determined as capable of such binding.

1001491 Clause 25: The ligand of clause 24, wherein the ligand is an antibody or antibody fragment.

1001501 Clause 26:The ligand of any one of clauses 17 to 23, wherein the ligand comprises a nucleotide sequence that specifically hybridises to a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50% or an RNA transcript thereof; and/or the ligand comprises a nucleotide sequence that comprises at least 10 contiguous nucleotides of a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50% or is an antisense sequence thereof.

100T511 In an embodiment, the ligand comprises a nucleotide sequence that comprises at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or 100 contiguous nucleotides ofa nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50% or is an antisense sequence thereof.

100t521 Clause 27: The ligand of any one of clauses T7 to 26, wherein the genome of said human comprises a nucleotide sequence having a cumulative human allele frequency of less than 50% and the sequence is found in at least 2 different ethnic populations (eg, found in at least 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19 or2O differenthuman ethnicpopulations (for example as per the populations in table 3)). In an example, numbers are with reference to the 1000 Genomes Project database.

1001 The ligand of any one of clauses 17 to 26, wherein the genome of said human comprises a nucleotide sequence having a cumulative human allele frequency of less than 50% and the sequence is found in at least 20 individuals distributed across at least 2 said different ethnic populations (eg, found in at least in at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150 individuals distributed across such many different ethnic populations). In an example, numbers are with reference to the 1000 Genomes Project database.

1001541 Clause 28: A pharmaceutical composition or kit for treating or preventing a condition or disease mediated by a TOT as recited in any preceding clause, the composition or kit comprising a ligand of any one of clauses 17 to 27; and optionally in combination with a label or instructions for use to treat and/or prevent said disease or condition in a human; optionally wherein the label or instructions comprise a marketing authorisation number (eg, an FDA or EMA authorisation number); optionally wherein the kit comprises an injection pen or TV container that comprises the ligand.

1001551 In an example, the label or instructions cover or describe use for a human comprising a TOl variant encoded by a nucleotide sequence as recited in clause 17.

1001561 Clause 29: A method of producing an anti-human TOl antibody binding site, the method comprising obtaining a plurality of anti-TOl antibody binding sites, screening the antibody binding sites for binding to a TOt comprising an amino acid sequence encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, or to a peptide thereof that comprises an amino acid variation from the corresponding sequence encoded by the TOl-encoding nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency, and isolating an antibody binding site that binds in the screening step.

1001571 Tn an embodiment of any aspect herein, the antibody, fragment or binding site is recombinant.

1001581 Tn the alternative, clause 29 provides: A method of producing an anti-human TOT antibody, the method comprising immunising a non-human vertebrate (eg, a mouse or a rat) with a TOT comprising an amino acid sequence encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, or to a peptide thereof that comprises an amino acid variation from the corresponding sequence encoded by the TOT-encoding nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency, and isolating an antibody that binds a TOT comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency ofess than 50% and/or a total human genotype frequency of less than 50%, and optionally producing a TOL-binding fragment or derivative of the isolated antibody.

1001591 The term "isohited" with reference to a ligand, antibody or protein, for example in any aspect, configuration, example or emodiment, means that a subject ligand, antibody, protein etc (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature.

Typically, an "isolated" ligand, antibody, protein etc constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode such an isolated ligand, antibody protein etc. Preferably, the isolated ligand, antibody protein etc is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.

1001601 For example, an "isolated" antibody is one that has been identified, separated and/or recovered from a component of its production environment (eg, naturally or recombinantly). Preferably, the isolated polypeptide is free of association with all other components from its production environment, eg, so that the antibody has been isolated to an FDA-approvabl e or approved standard. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least t5 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain, Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.

1001611 Imontitocoiqugates 1001621 The invention encompasses the ligand (eg, antibody) conjugated to a therapeutic moiety ("immunoconjugate"), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxin agents include any agent that is detrimental to cells. Examples of suitable cytotoxin agents and chemotherapeutic agents for forming immunoconjugates are known in the art, see for example, WO 05/103081.

[001631 13/specifics [001641 The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific mAbs may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al. (1991) J. Immunol. 147:60-69. The human anti-TOT (eg, anti-PCSK9) mAbs can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or a multispecific antibody th a second binding specificity.

[001651 An exemplary bi-specific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second CH3 include: DI6E, LI8M, N44S, KS2N, V57M, and V821 (by TIMGT; D356E, L358M, N384S, K392N, V397M, and V4221by EU) in the case of IgGI antibodies; N44S, KS2N, and V821 (IMGT; N384S, K392N, and V4221 by EU) in the case of IgG2 antibodies; and QTSR, N44S, KS2N, VS7M, R69K, E79Q, and V821 (by IN'IGT; Q3SSR, N3845, K392N, V397M, R409K, E419Q, and V4221 by EU) in the case of IgG4 antibodies, Variations on the bi-specific antibody format described above are contemplated within the scope of the present invention.

[00t661 Clause 30: The method of clause 29, comprising the step of obtaining a nucleic acid encoding the antibody, fragment, derivative or binding site and optionally inserting the nucleic acid in an expression vector.

[00t671 Clause 31: A kit for TOl genotyping a human, wherein the kit comprises a nucleic acid comprising a nucleotide sequence that specifically hybridises to a TOT nucleotide sequence selected having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or an RNA transcript thereof; and/or the nucleic acid comprises a nucleotide sequence that comprises at least 10 (eg, at least 10, 11, 12, 13, 14, 15,20,25,30,35,40,45,50 or 100) contiguous nucleotides of a 101 nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or is an antisense sequence thereof 1001681 For example, the nucleic acid hybridises to a region immediately flanking a nucleotide that is variant compared to the corresponding nucleotide of the TOl nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency. In an example, the nucleic acid hybridises to at two or more such variant nucleotides.

1001691 Specific hybridisation is under stringent conditions, as will be apparent to the skilled person, eg, conditions ofSxSSC, SxDenhardt's reagent, and 0.5% SDS at 650 C. Fool 701 Clause 32: A kit for TOL genotyping or phenotyping a human, wherein the kit comprises a ligand according to any one of clauses 17 to 27 or an antibody, fragment or derivative produced by the method of any one of clauses 29 to 3 1.

1001711 For example, the ligand specifically binds to an epitope comprising an amino acid that is variant compared to the corresponding amino acid of the TOl encoded by a nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency. h an example, the ligand specifically binds to an epitope comprising two or more such variant amino acids. In an example, specific binding means binding with an affinity (Kd) of 1mM, lOOnM, lOnM or mM or less, eg, as determined by SPR.

1001721 The tenn "epitope" is a region of an antigen that is bound by an antibody.

Epitopes may be defined as structural or functional, Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction, Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics, 1001731 Clause 33: Use of an anti-TOl ligand that binds a human 101 comprising an amino acid sequence encoded by a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, in the manufacture of a medicament for treating and/or preventing a TOT-mediated disease or condition in a human whose genome comprises a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

1001741 Clause 34: Use of an anti-TOl ligand that binds a human 101 comprising an amino acid sequence encoded by a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, in the manufacture of a medicament for targeting said TOT in a human to treat and/or prevent a disease or condition mediated by TOt.

1001751 The use of clause 33 or 34, wherein the ligand, human, disease or condition is according to any one of clauses ito 27.

1001761 Clause 35: A method of targeting a TOT for treating and/or preventing a TOT-mediated disease or condition in a human, the method comprising administering an anti-TOl ligand to a human comprising a TOt nucleotide sequence selected having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, whereby a TOT encoded by said nucleotide sequence is targeted.

1001771 Clause 36: The method of clause 35, wherein the method comprises targeting a human 101 comprising an amino acid sequence with said ligand to treat and/or prevent said disease or condition in said human, wherein said amino acid sequence is encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%.

1001781 Clause 37: A method of TOT genotyping a nucleic acid sample of a human, the method comprising identifying in the sample the presence of a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%, 1001791 Tn an example, the method comprises obtaining a TOT nucleic acid sample from the human and then carrying out the identifying step.

1001801 Clause 38: A method of TOt typing a protein sample of a human, the method comprising identifying in the sample the presence of a TOT amino acid sequence encoded by a TOT nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

1001811 In an example, the method comprises obtaining a TOt protein sample from the human and then carrying out the identifying step.

1001821 Clause 39: The method of clause 37 or 38, comprising obtaining a sample of semm, blood, faeces, hair, urine or saliva from a human, whereby the nucleic acid or protein sample is obtained for use in the step of identifying said sequence.

1001831 Clause 40: The method of any one of clauses 37 to 39, comprising using a ligand according to any one of clauses 17 to 27 to carry out said identifying step.

1001841 Clause 41: A diagnostic kit comprising a ligand that is capable of binding a human TOl comprising an amino acid sequence encoded by a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% and instructions for carrying out the method of clause 38 or 39, 100t851 Clause 42: A diagnostic kit comprising a nucleic acid probe comprising a nucleotide sequence that specifically hybridises a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50?/ or an RNA transcript thereof and instructions for carrying out the method of clause 38 or 39.

1001861 Clause 43: The method, ligand, composition, kit or use of any preceding clause, wherein the TOl is encoded by a nucleotide sequence having a cumulative human allele frequency from Ito 10% and/or a total human genotype frequency from I to about 5% or from Ito 15%.

1001871 Clause 44: The method, ligand, composition, kit or use of any preceding clause wherein the TOL is a human TOl selected from Table 4; optionally for treating and/or preventing a corresponding disease or condition as set out in Table 4.

1001881 For example, the TOl is human PCSK9, eg, a mature, cleaved, autocatalysed or active PCSK9. In an example, the disease is a cardiovascular disease such as hyperlipidaemia.

1001891 Ligands of the invention are useful, for instance, in specific binding assays, for genotyping or phenotyping humans, affinity purification of the TOT and in screening assays to identify other antagonists of TOT activity. Some of the ligands of the invention are useful for inhibiting binding of TOT to a congnate human receptor or protein, or inhibiting TOI-mediated activities.

100t901 The invention encompasses anti-TOT (eg, PCSK9) antibody ligands having a modified glycosylation pattern. In some applications, modification to remove undesirable gl ycosylation sites may be useful, or e.g., removal of a fucose moiety to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et a]. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

1001911 In an example, the invention features a pharmaceutical composition comprising a ligand of the invention, wherein the ligand is or comprises a recombinant human antibody or fragment thereof which specifically binds the TOt (eg, a rare variant as described herein) and a pharmaceutically acceptable carrier. In one embodiment, the invention features a composition which is a combination of an antibody ligand or antigen-binding fragment of an antibody of the invention, and a second therapeutic agent. The second therapeutic agent may be any of an anti-inflammatory agent, an anti-angiogenesis agent, a painkiller, a diuretic, a chemotherapeutic agent, an anti-neoplastic agent, a vasodilator, a vasoconstrictor, a statin, a beta blocker, a nutrient, an adjuvant, an anti-obesity agent and an anti-diabetes agent.

1001921 "Pharmaceutically acceptable" refers to approved or approvable by a regulatory agency of the USA Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. A "pharmaceutically acceptable carrier, excipient, or adjuvant" refers to an carrier, excipient, or adjuvant that can be administered to a subject, together with an agent, e.g., any antibody or antibody chain described herein, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

1001931 Tn an example, the invention features a method for inhibiting TOT activity using the anti-TOl ligand of the invention (eg, an antibody or antigen-binding portion of the antibody of the invention), wherein the therapeutic method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the ligand. The disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition or reduction of TOI activity.

1001941 By the phrase "therapeutically effective amount" is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (t999) The Art, Science and Technology of Pharmaceutical Compounding).

1001951 The term "specifically binds," or the like, means that a ligand, eg, an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about >< I O M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like, An isolated antibody that specifically binds a human TO! may, however, exhibit cross- reactivity to other antigens such as a TOT molecule from another species. Moreover, multi-specific antibodies (e.g., bispecifics) that bind to human TOI and one or more additional antigens are nonetheless considered antibodies that "specifically bind" TOT, as used herein.

Fool 961 Genotyping & Phenolyping 1001971 The skilled person will be familiar with techniques that can be used for accurate genotyping and application to the invention. These include the following.

1 Hybridization-based methods 11 Dynamic allele-specific hybridization 1,2 Molecular beacons 1.3 SNP microarrays 2 Enzyme-based methods 2.! Restriction fragment length polymorphism 2.2 PCR-based methods 2.3 Flap endonuclease 2.4 Primer extension 2,5 5'-nuclease 2.6 Oligonucleotide Ligation Assay 3 Other post-amplification methods based on physical properties of DNA 3,! Single strand conformation polymorphism 3,2 Temperature gradient gel electrophoresis 3.3 Denaturing high performance liquid chromatography 3,4 High-resolution melting of the entire amplicon 3.5 Use of DNA mismatch-binding proteins 3.6 SNP1ex (SNPlexTh' is a proprietary genotyping platform sold by Applied Biosystems).

1001981 Next-generation sequencing technologies such as pyrosequencing is also useful.

1001991 Reference is also made to GB2444410A and the genotyping method disclosed therein, which is incorporated herein by reference in its entirety, 1002001 Miniaturized assays, such as microarrays with oligonucleotide reagents immobilized on small surfaces, are frequently proposed for large-scale mutation analysis and high-throughput genotyping (Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome (Wang DG, Fan JB, Siao CJ, Bemo A, Young P, Sapoisky R, Ohandour 0, Perkins N, Winchester B, Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann NI, Morris MS, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen 5, Hudson Ti, Lipshutz R, Chee NI, Lander ES, Science.

1998 May 15; 280(5366): 1077-82). Other high-throughput methods discriminate alleles by differential hybridization, primer extension, ligation and cleavage of an allele-specific probe (Review Accessing genetic variation: genotyping single nucleotide polymorphisms, Syvanen AC, Nat Rev Genet, 200] Dec; 20 2):930-42; Review Techniques patents for SNP genotyping, Twyman RM, Primrose SB, Pharmacogenomics. 2003 ian; 4(l):67-79).

[002011 An approach for a fully automated, large-scale SNP analysis is the homogeneous' assay, i.e. a single-phase assay without separation steps, permitting continual monitoring during amplification. The TaqMan'M assay (Applied Biosystems), originally designed for quantitative real4ime PCR, is a homogeneous, single-step assay also used in determination of mutation status of DNA (see, eg, A.A. Komar (ed), Single Nucleotide Polymorphisms, Methods in Molecular Biology 578, DOl 10.1007/978-t-60327-4 t t-1t9, Humana Press, a part of Springer Science+Business Media, LLC; and Single Nucleotide Polymorphisms, Methods in Molecular BiologyTM Volume 578, 2009, pp 293-306, The TaqMan Method for SNP Genotyping, Gong-Qing Shen ef a!). The TaqMa.n SN]? Genotyping Assay exploits the 5'-exonuclease activity of AmpliTaq Gold DNA polymerase to cleave a doubly labeled probe hybridized to the SNP-containing sequence of ssDNA. Cleavage separates a 5'-fluorophore from a 3'-quencher leading to detectable fluorescent signal. The use of two allele-specific probes carrying different fluorophores permits SNP determination in the same tube without any post-PCR processing. Genotype is determined from the ratio of intensities of the two fluorescent probes at the end of amplification. Thus, rather than taking advantage of the full set of real-time PCR data as in quantitative studies, only end-point data are used.

[002021 TaqMan SNP genotyping in a high-throughput, automated manner is facilitated by the use of validated Pre-made TaqMan Genotyping assays, but Custom TaqMan Assays may also be used (High-throughput genotyping with single nucleotide polymorphisms, Ranade K, Chang MS, Ting CT, Pei D, Hsiao CF, Olivier NI, Pesich R, Hebert J, Chen YB, Dzau VJ, Curb D, Olshen R, Risch N, Cox DR, Botstein D, Genome Res, 200t Jul; 11(7):] 262-8; Assessment of two flexible and compatible SNP genotyping platforms: TaqMan SINP Genotyping Assays and the SNPIex Genotyping System, Dc Ia Vega FM, Lazaruk KD, Rhodes MD, Wenz MH, Mutat Res. 2005 Jun 3; 5 73(1-2): 111-35). The results of the assay can be automatically determined by genotyping software provided with real-time thermal cyclers (e.g. IQ software of Bio-Rad, Sequence Detection Software of Applied Biosystems).

[002031 Single nucleotide polymorphisms (SNP5) can be determined using TaqManm' real-time PCR assays (Applied Biosystems) arid commercial software that assigns genotypes based on reporter probe signals at the end of amplification. An algorithm for automatic genotype caling of SNPs using the full course of TaqMan real-time data is available for use (A. Callegaro eta!., Nucleic Acids Res. 2006; 34(7): e56, Published online 2006 April 14.

doi: 0.l093/nar!gkll85, PMCID: PMC]440877). The algorithm isunique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare.

[002041 The skilled person will be familiar with techniques that can be used for accurate phenotyping and application to the invention, These include the use of amino acid sequencing of isolated target protein and comparison of sequences from different variants (eg, with the most common variant). An antibody that specifically and selectively binds in the area of a SNP under stringent conditions can also be used to identify a particular variant, In another method, the genotype is determined and a corresponding amino acid sequence (phenotype) determined, eg, by in s/i/co translation.

[002051 Therapeutic Administration and Formulations [002061 The invention provides therapeutic compositions comprising the anti-TOT ligand, eg, antibodies or antigen-binding fragments thereof of the present invention, The administration of therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa, These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTTNTTh), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) JPharm Sci Technol 52:238-3 11.

1002071 The dose may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When the ligand, eg, antibody, of the present invention is used for treating various conditions and diseases associated with the TOl in an adult patient, it is advantageous to intravenously administer the antibody of the present invention nonnally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted.

1002081 Various delivery systems are known and can be used to administer the ligand or pharmaceutical composition of the invention, for example a ligand provided by e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al, (1987) J. Biol. Chem, 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The ligand or composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local, 1002091 The ligand or pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see [anger (1990) Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

1002101 In certain situations, the ligand or pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton (1987) CRC Crit, Ref Biomed, Eng. 14:20 1), In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, [anger and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).

1002t11 The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known, For example, the injectable preparations maybe prepared, e.g., by dissolving, suspending or emulsifying the antibody or its sail described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)I, etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule. A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention, Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused, In a disposable pen delivery device, there is no replaceable cartridge, Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device.

Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded, [002 121 Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a ligand or pharmaceutical composition of the present invention.

Examples include, but certainly are not limited to AUTOPENTM (Owen Mumford, Inc., Woodstock, UK), DISETRONICTM pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/2STM pen, HUIVIALOGTM pen, HUMALIN 70/3OTM pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPENTMI, H and IH (Novo Nordisk, Copenhagen, Denmark), NOVOPEN J'UNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes, NJ.), OPTIPENTTM, OPTIPEN PROTM, OPTIIPEN STARLETTM, and OPTICL1KT" (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTARTM pen (sanofi-aventis), the FLEXPENTM (Novo Nordisk), and the KWIKPENTM (Eli Lilly).

[002131 Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the ligand(s). Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is prefered that the aforesaid antibody is contained in about 5 to about 100mg and in about 10 to about 250mg for the other dosage forms.

[002t41 Exemplary TOIs [002151 For example in any configuration, aspect, concept, example or configuration of the invention, the or each TOt is selected from the group consisting of ABCFI; ACVR]; ACVRIB; ACVR2; ACVR2B; ACVRLI; ADORA2A; Aggrecan; AGR2; AICDA; AWl; AIGI; AKAP1; AKAP2; AIYIH; amyloid-beta; AMIHR2; ANGPTt; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOCt; AR; Axl; AZGPI (zinc-a-glycoprotein); B7.t; B7.2; BAD; BAFF; BAG1; BAli; BCL2; BCLÔ; BDNF; BLNK; BLR1 (MDRIS); BIyS; BMPI; BMP2; BMP3B (GDFIO); BMP4; BMP6; BMP8; BMPR]A; BMPR]B; BMPR2; BPAGI (plectin); BRCA1; Cl9orflO (IL27w); C3; C4A; CS; CSR1; CANTt; CASPt; CASP4; CAVt; CB1; CCBP2(D6/JAB61); CCLt (1-309); CCLII (eotaxin); CCLII (MCP-4); CCL]S (MIP-id); CCLI6 (1-ICC-4); CCLI7(TARC); CCL]8 (PARC); CCLI9(MIP-3b); CCL2 (MCP-i); MCAF; CCL2O (MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC / SIC-i); CCL23 (MPIF-l); CCL24 (IVIPIF-2 I eotaxin-2); CCL2S (TECK); CCL26 (eotaxin- 3); CCL27 (ClACK /ILC); CCL28; CCL3 (MW-la); CCL4 MIP-ib); CCLS (RANTES); CCL7(MCP-3); CCL8 (mcp-2); CCNA]; CCNA2; CCNDI; CCNEI; CCNE2; CCRI (CKR1 / 11M145); CCR2 (mcp-1RB / RA);CCR3 (CKR3 / CMKBR3); CCR4; CCR5 (CMKBRS / ChemRt3); CCR6 (CMIKBR6 / CKR-L3 / STRL22 / DRY6); CCR7 (CICR7 / EBII); CCR8 (CMKBR8 / TEE.] / CKR-Lt); CCR9 (GPR-9-6); CCRLI (VSHKI); CCRL2 (L-CCR); CD7, CD164; CD19; CDtC; CD2O; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD4O; CD4OL; CD44; CD4SRB; CDS2; CD69; CD72; CD74; CD79A; CD79B; CD8; CD8O; CD8t; CD83; CD86; CD96; CD207; CDHI (E-cadherin); CDFIIO; CDHI2; CDHI3; CDHI8; CDFII9; CDFI20; CDFIS; CDFI7; CDH8; CDH9; CDK2; CDK3; CDK4; CDKS; CDK6; CDK7; CDK9; CDKN1A(p2lWapl/Cipl); CDKN1B (p27Kipl); CDKNIC; CDKN2A (pl6lNK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CELSR3; CERt; CHGA; CHGB; Chitinase; CHIRNG; CHST1O; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSFG; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLRI; CMIKOR1 (RDC1); CNIRI; COL18A1; COLIAI; COL4A3; COL6A1; CR2; CRP; CSFI (M-CSF); CRLF2; CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNBI (b-catenin); CTSB (cathepsinB); CX3CL1 (SCYDi); CX3CRI (V28); CXCR6; CXCL1 (GROI); CXCLIO (IP-lO); CXCLI 1 (1-TAC / IP-9); CXCLI2 (SDFI); CXCLJ3; CXCL]4; CXCLI6; CXCL2 (CR02); CXCL3 (CR03); CXCLS (ENA- 78 I LIX); CXCLo (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR ISTRL33 I Bonzo); CYBS; CYC]; CYSLTR]; DAB2IP; DAND5; DES; DKFZp45IJOI 18; DNCL; DPP4; E2F]; ECGF]; EDC]; EFNAI; EFNA3; EFNB2; EGE; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EphA4; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESRI; ESR2; F3 (TF); FADD; FasL; FASN; FCERIA; FCER2; FCGR3A; FCRL4; FGF; FOFI (aFGF); FGFIO; FGF] ; FGF]2; FGFI2B; FGF]3; FGII4; FGFIÔ; FGF]7; FGFI8; FGFI9; FGF2 (bEeF); FGF2O; FGF2I; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KOF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FRI (EPSILON); FR I (ZETA); FL112584; FLJ2SS3O; FLRTI (fibronectin); FLTI; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB 1; GAGEC I; Galectin-3; GALNAC4S-65T; GATA3; GDF5; CEll; CCII; CHR; GM-CSF; CNASI; GNRH; GPR2 (CCR1O); GPR3I; GPR44; GPR8I (FKSG8O); 0PR87; GPRI37C; GRCCIO(Ci0); GRP; GSN(Gelsolin); GSTPI; HAYCRI; HAVCR2; HDAC4; EDACS; FIDAC7A; HDAC9; hepcidin; hemojuvelin; HGE; HIFIA; HIP]; histamine and histamine receptors; ElLA-A; HLA-DRA; 11M74; HMOX1; H[JMCYT2A; ICEBERG; ICOS; 1D2; IFN-a; WNA1; IFNA2; IFNA4; IFNAS; IFNA6; IFNA7; IFNBI; IFNgamma; TFNWI; IGBP1; IGFI; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-i; RiO; ILIORA; IL1ORB; ff11; ILl IRA; IL-12; ILI2A; ILI2B; ILI2RBI; IL]2RB2; 1L13; ILI3RAI; ILHRA2; IEJ4; IL]5; IL]5RA; 1L16; Ll7; 11L17B; 11L17C; IL17R; 1L18; IL18BP; IL18R1; IL18RAP; 1L19; ILIA; ILIB; IL1F1O; ILIFS; ILIF6; ILIF7; IL1F8; IL1F9; ILIHY1; ILIR1; ILIR2; ILIRAP; ILIRAPLI; ILIRAPL2; IL]RL];ILIRL2 ILIRN; ]L2; ]L20; IL2ORA; lL2R; L22; ]L22R; ]L22RA2; 1L23; 1L24; 1L25; 1L26; 1L27; 1L28A; 1L28B; iL29; IL2RA; IL2RB; IL2RG; 1L3; 1L30; IIL3RA; 1L4; IL4R; 1LS; IL5RA; 1L6; ff6 receptor; IL6ST g1ycoprotein 130); 1L7; TL7R; 1L8; IL8RA; IL8RB; IL8RB; 1L9; IL9R; ILK; INHA; INHBA; 1NSL3; 1NSL4; IRAKI; IRAK2; ITGA]; ITGA2; TGA3; ITCA6 (a6 integrin); ITCAV; ITCB3; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JLfM; K6HF; KAII; KDR; MTLG; KLFS (GC Box BP); KLF6; KLK1O; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLKS; KLK6; KLK9; KRTI; KRTI9 (Keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); LAG3; LAMA5; LEP (leptin); LIGHT; Lingo-p75; Lingo-Troy; [PS; LRP5; LTA (TNF-b); [TB; LTB4R (OPR16); LTB4R2; LTBR; MACMARCKS; MAO or Omgp; MAP2K7 (c-Jun); TvIDK; MIBI; midkine; MW; MIP-2; MK167 (ICi-67); MMP2; MIvIP9; MS4A1; MSN'IB; MT3 (metallothionectin-ifi); MTSS 1; MUC I (mucin); MYC; MYD88; NCK2; neurocan; NaI.7; Nai,8; NFKB 1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NMEI (NM23A); NOX5; NPPB; NROBI; NROB2; NR]D1; NR]D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NIR2C1; NR2C2; NIR2E1; NR2E3; NIR2F1; NR2F2; NR2F6; NR3C]; NR3C2; NR4A]; NR4A2; NR4A3; NRSAI; NRSA2; NRÔA]; NRP]; NRP2; NTSE; NTN4; ODZI; OPG; OPRD]; OX4OL; 0X40; P2RX7; PAP; PARTI; PATE; PAWR; PCA3; PCNA; PCSK9, PD-i, PD-Li; PDGFA; PDGFB; PECAMi; PF4 (CXCL4); PGF; PGR; phosphacan; PLkS2; Placental Growth Factor (PIGF); PIK3CG; PLAU (uPA); PLG; PLXDCI; PPBP (CXCL7); PPID; PR]; PRKCQ; PRKDJ; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p2lRac2); RARB; RGSi; RGS13; RGS3; RNF11O (ZNF144); ROBO2; RORI; S100A2; SCGB1D2(lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 mammaglobin i); SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPiNA1; SERPINIA3; SERPINB5 (maspin); SERPINEI (PAT-i); SERPINFI; SHBG; SLA2; SLC2A2; SLC33AI; SLC43AI; SLIT2; SPPi; SPRRiB (Spri); ST6GALI; STABI; STAT6; STEAP; STEAP2; TB4R2; TBX2i; TCP1O; TDGFI; TEK; TGFA; TGFBI; TGFB1Ii; TGFB2; TGFB3; TGFBI; TGFBRI; TGFBR2; TGFBR3; TI-I IL; THBS] (thrombospondin-); THBS2; THBS4; THPO; TIE (Tie-i); TIM3; TMP3; tissue factor; TLRIO; TLR2; TLR3; TLR4; TLRS; TLRÔ; TLR7; TLR8; TLR9; TMPRSS6; TNT; TNT-a; TNFAIP2 (B94); TNFAIP3; TNFRSF1 IA; TNFRSF1A; TNIFRSF1B; TNFRSF21; TNFRSFS; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNIFRSF9; TNFSFIO (TRAIL); TNFSFI I (TRANCE); TNFSFI2(APO3L); TNFSF]3 (April); TNT SFi3B; TNFSFi4 (HVEM-L); TNIFSF1 5 (VEGI); TNFSFi 8; TNFSF4 (0X40 ligand); TNIFSFS (CD4O ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand; TNFSF8 (CD3O ligand); TNFSF9 (4-] BB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase ha); TP53; TPMi; TPM2; TRADD; TRAFI; TRAF2; TRAF3; TRAF4; TRAFS; TRAF6; TRAIL; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGFA; VEGFB; VEGFC; versican; VEIL CS; VLA-4; Wnt7A; XCL1 (lymphotactin); XCL2 (SCM-lb); XCRI (GPRS / CCXCR1); YYI; and ZFPM2.

[002161 In an example, the T0I is a human 101 selected from Table 4.

[002171 In an example, the TOI is 0X40 ligand.

[002181 lii an example, the TOI is 0X40, 1002191 In an example, the TOt is PCSK9.

1002201 In an example, the TOT is IL6 receptor.

1002211 In an example, the TOT is LIGHT.

1002221 In an example, the TOT is VEGF-A.

1002231 In an example, the TOT is TNF alpha, 1002241 In an example, the TOt is PIGE.

1002251 In an example, the TOT is IGFTR.

1002261 In an example, the TOt is OPG.

1002271 In an example, the TOt is NGF.

1002281 In an example, the TOT is BMPo.

1002291 In an example, the TOT is hemojuvelin.

1002301 In an example, the TOt is VEGE receptor, 1002311 In an example, the TOt is PDGF receptor, 1002321 In an example, the TOT is stem cell factor receptor, 1002331 In an example, the TOT is hepcidin.

1002341 In an example, the TOT is IL-4 receptor alpha, 1002351 In an example, the TOt is sclerostin.

1002361 In an example, the TOT is IL-13 receptor.

1002371 In an example, the TOT is CD7.

1002381 In an example, the TOt is delta-like ligand-4 (D114).

1002391 In an example, the TOT is HGF.

1002401 In an example, the TOT is angiopoietin-2 (Ang2).

1002411 In an example, the TOT is GDF8.

1002421 In an example, the TOt is ERBB3.

1002431 In an example, the TOT is IL-17 receptor.

1002441 In an example, the TOT is CD4O.

1002451 In an example, the TOt is CD4O ligand.

1002461 In an example, the TOT is EGFR.

1002471 For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited on'y by the claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

1002481 For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

1002491 The terms "decrease", "reduced", or "reduction" are all used herein to mean a decrease by a statistically significant amount. In some embodiments, "reduce," "reduction" or "decrease" typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, "reduction" does not encompass a complete reduction as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder. However, for example, for the purposes of lowering or reducing cholesterol level, for example, a reduction by about 5-10 points can be considered a "decrease" or "reduction." 1002501 In certain aspects of all embodiments of the invention, the term "inhibition" is used. Inhibition refers and refers to decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more including 100% inhibition as compared to a reference level. "Complete inhibition" refers to a 100% inhibition as compared to a reference level.

1002511 The terms "increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms "increased", "increase", "enhance", or "activate" can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between tO-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

1002521 As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For the removal of doubt, "substantially" can refer to at least a 90% extent or degree of a characteristic or property of interest, e.g. at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or greater.

[002531 As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus, Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters, In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, "individual," "patient" and "subject" are used interchangeably herein. In some embodiments, the subject can be a non-human vertebrate, e.g. a primate, a rodent, a mouse, a rat, a pig, a sheep, a zebrafish, a frog, etc. [002541 Preferably, the subject is a mammal, The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples, Mammals other than humans can be advantageously used as subj ects that represent animal models of a disease or condition, e.g., a cardiovascular condition. A subject can be male or female.

[002551 A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition, For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.

[002561 A "subject in need" or "human in need" of treatment for a particular condition can be a subject having that condition, such as increased cholesterol levels, diagnosed as having that condition, or at risk of developing that condition.

[002571 As used herein, the terms "protein" arid "polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids th natural amino acids. When referring to "modified polypeptides" one refers to polypeptides that include modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or frmnction, "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins with the specified sequence. One can also use peptide homologs, peptide orthologs, peptide paralogs, peptide fragments and other equivalents, variants, fragments, and analogs of the peptides as these terms are understood by one of ordinary skill in the art.

[002581 As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid. The nucleic acid can be either single-stranded or double-stranded, A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA. In some aspects one can also use analogs of nucleic acids.

[002591 As used herein, the term "nucleic acid probe" refers to an isolated oligonucleotide molecule having a nucleic acid sequence which can hybridize to a target nucleic acid sequence, e.g. specifically hybridize to the target sequence. In some embodiments, a nucleic acid probe can further comprise a detectable label. In some embodiments, a nucleic acid probe can be attached to a solid surface, In some embodiments, a nucleic acid from is from about 5 nt to about 100 nt in length.

[002601 As used herein, the term "siRNA" refers to a nucleic acid that forms an RNA molecule comprising two individual strands of RNA which are substantially complementary to each other, Typically, the siRNA is at least about 15-40 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is about 5-40 nucleotides in length, and the double stranded siRNA is about 15-40 base pairs in length, preferably about 19-25 base nucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides in lengthy In some embodiments, a siRNA can be blunt-ended, In some embodiments, a siRNA can comprise a 3' and!or 5' overhang on each strand having a length of about 0, 1, 2, 3,4, or 5 nucleotides, The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand.

The siRNA molecules can also comprise a 3' hydroxyl group. In some embodiments, the siRNA can comprise aS' phosphate group. A siRNA has the ability to reduce or inhibit expression of a gene or target RNA when the siRNA is present or expressed in the same cell as the target gene, e.g, the target RNA. siRNA-dependent post-transcriptional silencing of gene expression involves cutting the target RNA molecule at a site guided by the siRNA, 1002611 As used herein, "PCSK9" or"proprotein convertase subtilisin/kexin type 9" refers to a serine protease involved in regulating the levels of the low density lipoprotein receptor (LDLR) protein (Horton et al., 2007; Seidah and Prat, 2007). PCSK9 has been shown to directly interact with the LDLR protein, be endocytosed along with the LDLR, and co-immunofluoresce with the LDLR throughout the endosomal pathway (Lagace et al., 2006).

PCSK9 is a prohormone-proprotein convertase in the subtilisin (58) family of serine proteases (Seidah et al., 2003). The sequence of PCSK9 for a variety of species is known, e.g., human PCSK9 (NCBI Gene ID No: 255738). Nucleotide and polypeptide sequences for a number of PCSK9 isoforms are provided herein, e.g., SEQ ID NOs: 1-3 7.

1002621 PCSK9 exists as both a pro-form and a mature form, Autocatalysis of the PCSK9 proform occurs between G1n152 and Ser153 (VFAQSiP) Naureckiene et al., 2003), and has been shown to be required for its secretion from cells (Seidah et al,, 2003), The inactive form prior to this cleavage can be referred to herein as the "inactive", "pro-form", or "unprocessed" form ofPCSK9. The C-terminal fragment generated by the autocatalysis event can be referred to herein as the "mature," "cleaved", "processed" or "active" PCSK9.

Examples of pro-form and mature PCSK9 isoforms are provided herein, see, e.g. SEQ ID NOs: 1-27.

1002631 As used herein, the "catalytic domain" of PCSK9 refers to the portion of a PCSK9 polypeptide corresponding to positions 153 to 449 of PCSK9, e.g. of SEQ ID NO: I. As used herein, the "C-terminal domain" of PCSK9 refers to the portion of a PCSK9 polypeptide corresponding to positions 45 0-692 of PC SK9, e.g., of SEQ ID NO: 1.

1002641 As used herein, a disease or condition "mediated by PCSK9" refers to a disease or condition which is caused by or characterized by a change in PCSK9, e.g. a change in expression level, a change in activity, and/or the presence of a variant or mutation ofPCSK9.

Non-limiting examples of such diseases or conditions can include, for example, a lipid disorder, hyperlipoproteinemia, hyperlipidemia; dyslipidemia; hypercholesterolemia, a heart attack, a stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, claudication, type II diabetes, high blood pressure, and a cardiovascular disease or condition.

In an example, the disease or condition is an inflammatory or autoimmune disease or condition. Methods of identifying and/or diagnosing such diseases and conditions are well known to medical practitioners of ordinary skill.

1002651 A subject at risk of having or developing a disease or condition mediated by PCSK9 can be a subject exhibiting one or more signs or symptoms of such a disease or condition or having one or more risk factors for such a disease or condition, e.g. being overweight, having elevated cholesterol level, comprising one or more genetic polymorphisms known to predispose to the disease or condition, e.g., elevated cholesterol level, such as having a mutation in the LDLR (encoding low-density lipoprotein receptor) or APOB (encoding apolipoprotein B) or in the PCSK9 gene and/or having a family history of such a disease or condition.

1002661 As used herein, "ligand" refers to a molecule which can bind, e.g., specifically bind, to a second molecule or receptor. In some embodiments, a ligand can be, e.g., an antibody, antibody fragment, antibody portion, and/or affibody.

1002671 The term "variant" as used herein refers to a peptide or nucleic acid that differs from the polypeptide or nucleic acid (eg, the most common one in humans, eg, most frequent in a database as disclosed herein, such as the 1000 Genomes Project database) by one or more amino acid or nucleic acid deletions, additions, yet retains one or more specific functions or biological activities of the naturally occurring molecule, Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring amino acid residue. Such substitutions may be classified as "conservative", in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size, Such conservative substitutions are well known in the art, Substitutions encompassed by the present invention may also be "non-conservative", in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (e.g., substituting a charged or hydrophobic amino; acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid. In some embodiments amino acid substitutions are conservative. Also encompassed within the term variant when used with reference to a polynucleotide or polypeptide, refers to a polynucleotide or polypeptide that can vary in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild-type polynucleotide or polypeptide).

1002681 Variants of PCSK9 are provided elsewhere herein, Variants of PCSK9 can include the forms described herein as a, f c, r, p, m, e h, aj, and q. Sequences of these variants are provided herein, see, e.g, SEQ ID NOs:1-27 and in Table 1.

1002691 lii some aspects, one can use "synthetic variants", "recombinant variants", or "chemically modified" polynucleotide variants or polypeptide variants isolated or generated using methods well known in the art, "Modified variants" can include conservative or non-conservative amino acid changes, as described below, Polynucleotide changes can result in amino acid substitutions, additions, deletions, frisions and tmncations in the polypeptide encoded by the reference sequence, Some aspects use include insertion variants, deletion variants or substituted variants with substitutions of amino acids, including insertions and substitutions of amino acids and other molecules) that do not normally occur in the peptide sequence that is the basis of the variant, for example but not limited to insertion of ornithine which do not normally occur in human proteins, The term "conservative substitution," when describing a polypeptide, refers to a change in the amino acid composition of the polypeptide that does not substantially alter the polypeptide's activity. For example, a conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties, Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine, 1002701 "Conservative amino acid substitutions" result from replacing one amino acid with another having similar stmctural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine, Thus, a "conservative substitution" of a particular amino acid sequence refers to substitution of those amino acids that are not critical for polypeptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitution of even critical amino acids does not reduce the activity of the peptide, (i,e, the ability of the peptide to penetrate the blood brain barrier (BBB)). Conservative substitution tables providing frmnctionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Trvptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984), incorporated by reference in its entirety.) In some embodiments, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids can also be considered "conservative substitutions" if the change does not reduce the activity of the peptide.

Insertions or deletions are typically in the range of about Ito S amino acids. The choice of conservative amino acids may be selected based on the location of the amino acid to be substituted in the peptide, for example if the amino acid is on the exterior of the peptide and expose to solvents, or on the interior and not exposed to solvents.

[002711 In alternative embodiments, one can select the amino acid which will substitute an existing amino acid based on the location of the existing amino acid, i.e. its exposure to solvents (i.e. if the amino acid is exposed to solvents or is present on the outer surface of the peptide or polypeptide as compared to internally localized amino acids not exposed to solvents). Selection of such conservative amino acid substitutions are well known in the art, for example as disclosed in Dordo et al, J. Mol Biol, 1999, 2 7, 721-739 and Taylor et al, J. Theor. Biol. 1 19(1986);205-218 and S. French and B. Robson, J. Mol. Evol. 19(1983)171.

Accordingly, one can select conservative amino acid substitutions suitable for amino acids on the exterior of a protein or peptide (i.e. amino acids exposed to a solvent), for example, but not limited to, the following substitutions can be used: substitution of Y with F, T with S or K,PwithA,EwithDorQNwithDorG,RwithK,GwithNorA,TwithSorK,Dith N or E, I with L or V, F withY, S with T or A, R with K, U with N or A, K with R, A with S, K or P. [002721 In alternative embodiments, one can also select conservative amino acid substitutions encompassed suitable for amino acids on the interior of a protein or peptide, for example one can use suitable conservative substitutions for amino acids is on the interior of a protein or peptide (i.e. the amino acids are not exposed to a solvent), for example but not limited to, one can use the following conservative substitutions: where Y is substituted with F, T with A or 5, I with L or V, W withY, M with L, N with D, G with A, T with A or 5, D with N, I with L or V. F with Y or L, S with A or T and A with 5, U, T or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term of variants.

[002731 As used herein an "antibody" refers to IgG, 1gM, IgA, IgD or IgE molecules or antigen-specific antibody fragments thereof (including, but not limited to, a Fab, F(ab')2, Fv, disulphide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulphide-linked scfv, diabody), whether derived from any species that naturally produces an antibody, or created by recombinant DNA technology; whether isolated from semm, B-cells, hybridomas, transfectomas, yeast or bacteria. Antibodies can be humanized using routine technology.

[002741 As described herein, an "antigen" is a molecule that is bound by a binding site on an antibody agent. Typically, antigens are bouud by antibody ligands and are capable of raising an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof. The term "antigenic determinant" refers to an epitope on the antigen recognized by an antigen-binding molecule, and more particularly, by the antigen-binding site of said molecule.

[002751 As used herein, the term "antibody fragment" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody fragment can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments, an antibody fragment can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody fragment" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (see, e.g. de Wildt et al,, Fur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, 1gM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.

[002761 As used herein, "antibody variable domain" refers to the portions of the light and heavy chains of antibody molecules that include amino acid sequences of Complementarity Determining Regions (CDR5; ie,, CDR], CDR2, and CDR3), and Framework Regions (FR5), VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. According to the methods used in this invention, the amino acid positions assigned to CDRs and FRs may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)) or according to IMGT nomenclature.

[002771 D domain or region refers to the diversity domain or region of an antibody chain.

J domain or region refers to the joining domain or region of an antibody chain.

[002781 An antibody "gene segment", e.g. a VU gene segment, D gene segment, or il-I gene segment refers to oligonucleotide having a nucleic acid sequence that encodes that portion of an antibody, e.g. a VH gene segment is an oligonucleotide comprising a nucleic acid sequence that encodes a polypeptide VH domain.

[002791 The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" ("FR"). The extent of the framework region and CDRs has been precisely defined (see, LMGT or Kabat. E. A,, et al, (199]) Sequences of Proteins of Immunological Interest, Fifih Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al, (1987) J. Mol. Biol, 196:901-917; which are incorporated by reference herein in their entireties). Each VU and VL is typically composed of three CDRs and four Ui(s, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[002801 The terms "antigen-binding fragment" or "antigen-binding domain", which are used interchangeably herein are used to refer to one or more fragments of a full length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH I domains; (ii) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al,, (1989) Nature 341:544-546; which is incorporated by reference herein in its entirety), which consists of a VII or VL domain; and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality.

1002811 As used herein, the term "antibody binding site" refers to a polypeptide or domain that comprises one or more CDRs of an antibody and is capable of binding an antigen. For example, the polypeptide comprises a CDR3 (eg, HCDR3). For example the polypeptide comprises CDRs 1 and 2 (eg, HCDR1 and 2) or CDRs 1-3 of a variable domain of an antibody (eg, HCDR5I-3). In an example, the antibody binding site is provided by a single variable domain (eg, a VH or VL domain). In another example, the binding site comprises a VHIVL pair or two or more of such pairs.

1002821 As used herein, the term "specific binding" refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. For example, in an diagnostic test the specific binding of a ligand can distinguish between two variant PCSK9 proteins as described herein, In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity. In the context of oligonucleotide strands which interact via hybridization, specific binding can be "specific hybridization." 1002831 Additionally, and as described herein, a recombinant human(ized) antibody can be further optimized to decrease potential immunogenicity, while maintaining functional activity, for therapy in humans, In this regard, functional activity means a polypeptide capable of displaying one or more known frmnctional activities associated with a recombinant antibody or antibody reagent thereof as described herein. Such functional activities include, e,g, the ability to bind to a target molecule, 1002841 The term "immunizing" refers to the step or steps of administering one or more antigens to an animal so that antibodies can be raised in the animal Generally, immunizing comprises injecting the antigen or antigens into the animal. Immunization can involve one or more administrations of the antigen or antigens, Suitable methods are prime-boost and RIMMS procedures as known to the skilled person in the art, 1002851 As used herein, an "affibody" refers to a relatively small synthetic protein molecule that has high binding affinity for a target protein (e.g. for PCSK9 or a variant therefo), Affibodies are composed of a three-helix bundle domain derived from the IgG-binding domain of staphylococcal protein A, The protein domain consists of aSS amino acid sequence, with 13 randomized amino acids affording a range of affibody variants. Despite being significantly smaller than an antibody (an affibody weighs about 6 kDa while an antibody commonly weighs about 150 kDa), an affibody molecule works like an antibody since its binding site is approximately equivalent in surface area to the binding site of an antibody.

[002861 As used herein, "VH323*04" refers to a human Viii domain variant comprising the polypeptide sequence of SEQ ID NO: 38. As opposed to the reference sequence, VH3- 23 *04 has a valine residue instead of a leucine residue (see Figures 3 and 4; L24V, numbering including signal sequence; valine at position 5 shown in Figure 4) as a result of the presence of the rs56069819 SNP in the nucleic acid sequence encoding the \TH domain.

As used herein, "rs560698 19" refers to a mutation or variant in a VU gene segment from adenosine to cytosine (or thymine to guanine, depending upon the strand of DNA which is being read), resulting in the VH domain encoding VH323*04. Rs56069819 is depicted in Figure 4 and SEQ ID NO: 39, which demonstrate the T->G mutation (it is noted that the dbSNP entry for RS5606819 depicts the other strand, which comprises the A->C mutation).

Further description of VH323*04 can be found, e.g., in US Patent Publication 2013/007i405; which is incorporated by reference herein in its entirety.

100287] As used herein, "determine" or "determining" refers to ascertaining, e.g., by a quantitative or qualitative analysis. As used herein, "has been determined" can refer to ascertaining on the basis of previously obtained information or simultaneously obtained information, 100288] In some aspects of all embodiments of the invention selecting can include automation such as a computer implemented software program that upon input of the relevant data such as ethnicity or a panel of SNIP data can make the determination based on the instructions set forth herein, 100289] As used herein, "assaying" refers to assessing, evaluating, quantifying, measuring, or characterizing an analyte, e.g., measuring the level of an analyte in a sample, identifying an analyte, or detecting the presence or absence of an analyte in a sample. In some embodiments, assaying refers to detecting a presence or absence of the analyte of interest. In some embodiments, assaying refers to quantifying an amount of an analyte, e.g., providing a measure of concentration or degree of analyte abundance. in some embodiments, assaying refers to enumerating the number of molecules of analyte present in a sample and/or specimen, e.g., to determine an analyte copy number.

100290] As used herein "multiplex" refers to the carrying out of a method or process simultaneously and in the same reaction vessel on two or more, typically three or more, different target sequences, e.g. on two or more isoforms of PC SK9, or PCSK9 and an additional target.\ multiplex analysis typically includes analysis of 10-50; 10-100; iO- 1000, 10-5000, 10-10000 reactions in a multiplex format, such as a multiwa!!, an array, or a muidchannei reaction.

100291] Often the analysis or multiplex analysis is also automated using robotics and typically software executed by a computer and may include a robotic handling of samples, aulornatic or robotic selection of positive or negative results, assaying for presence of absence of a target, such as a nucleic acid polymorphism or a protein variant.

100292] The term "biological sample" or "test sample" as used herein denotes a sample taken or isolated from a biological organism, e.g., a sample from a subject.

Exemplary biological samples include, but are not limited to, a biofluid sample; semm; plasma; urine; saliva; hair, epithelial cells, skin, a tumor biopsy and/or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term "test sample" or "biological sample" also includes untreated or pretreated (or pre-processed) biological samples. For the analysis of nucleic acids, the biological sample should typically comprise at least one cell comprising nucleic acids.

100293] The test sample can be obtained by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells (e.g. isolated at a prior time point and isolated by the same or another person). In addition, the test sample can be freshly collected or a previously collected, refrigerated, frozen or otherwise preserved sample.

100294] In some embodiments, the test sample can be an untreated test sample. As used herein, the phrase "untreated test sample" refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution, Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof In some embodiments, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein, After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof In some embodiments, the test sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an expression product as described herein.

100295] As used herein, "genotyping" refers to a process of determining the specific allelic composition of a cell and/or subject at one or more position within the genome, e.g. by determining the nucleic acid sequence at that position. Genotyping refers to a nucleic acid analysis and/or analysis at the nucleic acid level. As used herein, "phenotyping" refers a process of determining the identity and/or composition of an expression product of a cell and/or subject, e.g. by detennining the polypeptide sequence of an expression product.

Phenotyping refers to a protein analysis and/or analysis at the protein level.

100296] As used herein, the term "nucleic acid amplification" refers to the production of additional copies of a nucleic acid sequence and is typically carried out using polymerase chain reaction (PCR) or ligase chain reaction (LCR) technologies well known in the art (Dieffenbach, C. W. and G, S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N. Y,), Other methods for amplification are also contemplated in aspects of the invention.

100297] The term "allele-specific amplification" refers to a reaction (e.g., PCR reaction) in which at least one of the primers (e.g., allele-specific primer) is chosen from a polymorphic area of gene (e.g., single nucleotide polymorphism), with the polymorphism located at or near the primer's 3-end, A mismatched primer will not initiate amplification, whereas a matched primer will initiate amplification. The appearance of an amplification product is indicative of the presence of the polymorphism.

100298] As used herein, "sequencing" refers to the determination of the exact order of nucleotide bases in a strand of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) or the exact order of amino acids residues or peptides in a protein. Nucleic acid sequencing can be done using Sanger sequencing or next-generation high-throughput sequencing.

100299] As used herein "next-generation sequencing" refers to oligonucleotide sequencing technologies that have the capacity to sequence oligonucleotides at speeds above those possible with conventional sequencing methods (e.g. Sanger sequencing), due to performing and reading out thousands to millions of sequencing reactions in parallel.

Non-limiting examples of next-generation sequencing methods/platforms include Massively Parallel Signature Sequencing (Lynx Therapeutics); 454 pyro-sequencing (454 Life Sciences! Roche Diagnostics); solid-phase, reversible dye-terminator sequencing (Solexallllumina): SOLiD technology (Applied Biosystems); Ion semiconductor sequencing (iON Torrent); DNA nanoball sequencing (Complete Genomics); and technologies available from Pacific Biosciences, Jntelligen Bio-systems, Oxford Nanopore Technologies, and Helicos Biosciences. Next-generation sequencing technologies and the constraints and design parameters of associated sequencing primers are well known in the art (see, e.g. Shendure, et al., "Next-generation DNA sequencing," Nature, 2008, vol. 26, No. 10, 1135- 1145; Mardis, "The impact of next-generation sequencing technology on genetics," Trends in Genetics, 2007, vol. 24, No. 3, pp. 133-141; Su, et al., "Next-generation sequencing and its applications in molecular diagnostics" Expert Rev Mol Diagn, 2011, 1 1(3):333-43; Zhang et al,, "The impact of next-generation sequencing on genomics", J Genet Genomics, 2011, 38(3):95-109; (Nyren, P. et al, Anal Biochem 208: 17175 (1993); Bentley, D. R. Curr Opin Genet Dcv 16:545-52 (2006); Strausberg, R. L., et al. Dmg Disc Today 13:569-77 (2008); U.S. Pat. No. 7,282,337; U.S. Pat. No. 7,279,563; U.S. Pat. No. 7,226,720; U.S. Pat. No, 7,220,549; U.S. Pat. No. 7,169,560; U.S. Pat, No. 6,818,395; U.S. Pat, No. 6,911,345; US Pub. Nos. 2006/0252077; 2007/0070349; and 20070070349; which are incorporated by referene herein in their entireties).

1003001 As used herein, "nucleic acid hybridization" refers to the pairing of complementary RNA and DNA strands as well as the pairing of complementary DNA single strands. In some embodiments, nucleic acid hybridization can refer to a method of determining a nucleic acid sequence and/or identity by hybridizing a nucleic acid sample with a probe, e.g. Northern or Southern blot analysis or microarray analysis.

1003011 As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes notjust the improvement of symptoms or markers, but also a cessation of or at least sI ong of progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (/.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment). For treatment to be effective a complete cure is not contemplated. The method can in certain aspects include cure as well.

100302] As used herein, the term "pharmaceutical composition" refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

100303] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subj ect.

100304] Multiple compositions can be administered separately or simultaneously.

Separate administration refers to the two compositions being administered at different times, e.g. at least 10, 20, 30, or 10-60 minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. One can also administer compositions at 24 hours apart, or even longer apart.

Alternatively, two or more compositions can be administered simultaneously, e.g. less than or less than 5 minutes apart. Compositions administered simultaneously can, in some aspects, be administered as a mixture, with or without similar or different time release mechanism for each of the components.

100305] As used herein, "contacting" refers to any suitable means for delivering, or exposing, an agent to at least one complex, enzyme, or cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art.

100306] As used herein, "obtain" refers to any method of acquiring, securing, procuring, or coming into the possession of, e.g. a sample. Obtaining a biological sample from a subject can comprise physical removing a sample from a subject (e.g. drawing blood or taking a hair or saliva sample) without or without active participation from the subject; receiving a sample from a subject (e.g. the subject collects a saliva or hair sample themselves and provides it, e.g. in a container provided for the purpose); or procuring a sample from a storage facility, medical facility, or medical provider. Obtain from the human or subject, refers to an active step of, e.g., drawing blood or taking a tissue or cell sample.

1003071 As used herein, "cholesterol level" refers to a level of one or more of total cholesterol, LDL cholesterol, HDL cholesterol, and/or triglycerides. Cholesterol levels can be the level of cholesterol in the blood of a subject.

1003081 As used herein in reference to cholesterol levels, "maintain" refers to preventing the level from worsening (e.g. increasing). In some embodiments, maintaining a particular level refers to a process that results in the cholesterol level not increasing by more than 10% overtime, Maintaining may also refer to maintaining a previously achieved level. For example, if a human has received statin treatment, one can maintain the cholesterol level achieved using the statin treatment.

1003091 In some embodiments, the subject treated according to the methods described herein has previously had their cholesterol level reduced, As used herein, "previously reduced" indicates that at a prior point in time, the subject experienced a decrease in cholesterol levels, The decrease can be due to administration of a pharmaceutical composition (e.g. administration of a composition as described herein or another composition, e.g. a statin) or due to another cause, e.g. a change in diet and/or exercise.

1003101 An existing treatment for high cholesterol levels is the administration of a statin, As referred to herein, a "statin" (also known as I-fvlG-CoA reductase inhibitors) are inhibitors of the enzyme HMG-coA reductase, which mediates cholesterol production in the liver. Statins, by competitively binding HMG-CoA reductase, prevent the binding of FIJVIG-CoA to the enzyme and thereby inhibit the activity of the reductase (e.g. the production of mevalonate). Non-limiting examples of statins can include atorvastatin (LIPITORTM), fluvastatin (LESCOLTM), lovastatin (MEVAC ORTM, A.LTOCORTM), pitavastatin (LIIVALOTM), pravastatin (PRAVACHOLTM), rosuvastatin (CRESTORTh9, and simvastatin (ZOCORTM). Statins can be administered in combination with other agents, e.g. the combination of ezetimibe and simvastatin, 1003111 Some subjects are, or become, resistant to statin treatment. As used herein, "resistant to statin treatment" or "reduced responsiveness to statin treatment" refers to a sub] ect exhibiting a statistically significantly lower response to the administration of a statin as compared to a reference level. The reference level can be, e.g., the average response for a population of subjects or the level of the individual subject at an earlier date. A response to statin treatment is readily measured by one of skill in the art, e.g., measurement of cholesterol levels, changes in cholesterol levels, and/or 1-IMG-CoA reductase activity.

1003121 As used herein, the term "detectable label" refers to a molecule or moiety that can be detected, e.g. measured and/or determined to be present or absent, Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g, antibodies and nucleic acid probes) are well known in the art, 1003131 In some embodiments, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means, The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies).

The detectable label can be linked by covalent or non-covalent means to the reagent, Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

1003141 In other embodiments, the detectable label can be a fluorescent compound, When the fluorescently label is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence, In some embodiments, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3Tht, Cy5TM, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5"M, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Greentm", rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes, 6-carboxythiorescein (commonly known by the abbreviations FAN'l and F), 6-carboxy-2',4',7',4,7-hexachlorofiuorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfiuorescein (JOE or J), N,N,N',N'-tetramethyl-ôcarboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or 05), 6-carboxyrhodamine-60 (R606 or Go), and rhodamine 110; cyanine dyes, e.g. Cy3, CyS and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, CyS, etc; BODIPY dyes and quinoline dyes. In some embodiments, a detectable label can be a radiolabel including, but not limited to 3H, 1251 %5, 4, 32P, and 33P. In some embodiments, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use as a detectable label can include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VT-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester, In some embodiments, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

1003151 Tn some embodiments, reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, CA. A reagent can also be detectably labeled using fluorescence emitting metals such as 12Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethyl enetri aminepentaacetic acid (DTPA) or ethyl enediaminetetraaceti c acid (EDTA).

1003161 As used herein, "authorization number" or "marketing authorization number" refers to a number issued by a regulatory agency upon that agency determining that a particular medical product and/or composition maybe marketed and/or offered for sate in the area under the agency's jurisdiction. As used herein "regulatory agency" refers to one of the agencies responsible for evaluating, eg, the safety and efficacy of a medical product and/or composition and controlling the sales/marketing of such products and/or compositions in a given area. The Food and Drug Administration (FDA) in the US and the European Medicines Agency (EPA) in Europe are but two examples of such regulatory agencies. Other non-limiting examples can include SDA, MPA, IvIIHIPRA, IIVIA, ANMAT, Hong Kong Department of Health-Dmg Office, CDSCO, Medsafe, and KFDA.

1003171 As used herein, "injection device" refers to a device that is designed for carrying out injections, an injection including the steps of temporarily fluidically coupling the injection device to a person's tissue, typically the subcutaneous tissue. An injection further includes administering an amount of liquid drug into the tissue and decoupling or removing the injection device from the tissue. In some embodiments, an injection device can be an intravenous device or IV device, which is a type ofinjection device used when the target tissue is the blood within the circulatory system, e.g., the blood in a vein. A common, but non-limiting example of an in] ection device is a needle and syringe.

1003181 As used herein, a "buffer" refers to a chemical agent that is able to absorb a certain quantity of acid or base without undergoing a strong variation in p11 1003191 As used herein, "packaging" refers to how the components are organized and/or restrained into a unit fit for distribution and/or use. Packaging can include, e.g., boxes, bags, syringes, ampoules, vials, tubes, clamshefl packaging, barriers and/or containers to maintain sterility, labeling, etc. 1003201 As used herein, "instructions" refers to a display of written, printed or graphic matter on the immediate container of an article, for example the written material display"ed on a vial containing a pharmaceutically active agent, or details on the composition and use of a product of interest included in a kit containing a composition of interest. Lnstmctions set forth the method of the treatment as contemplated to be administered or performed.

1003211 As used herein, a "solid surface" refers to an object suitable for the attachment of biomolecules. Non-limiting examples of a solid surface can include a particle (including, but not limited to an agarose or latex bead or particle or a magnetic particle), a bead, a nanoparticle, a polymer, a substrate, a slide, a coverslip, a plate, a dish, a well, a membrane, and/or a grating. The solid surface can include many different materials including, but not limited to, polymers, plastics, resins, polysaccharides, silicon or silica based materials, carbon, metals, inorganic glasses, and membranes, 1003221 As used herein, "classification" of a subject, e.g., classification of the subject's ancestry refers to determining if the subject has biological ancestors who originated in a particular geographical area, and are therefore likely to have particular genetic variants found in the populations which have historically occupied that area. Classification can comprise, e.g. obtaining information on the subject's family, interviewing the subject or a family member regarding their biological family's ancestry, and/or genetic testing.

Classification can be on the basis used for the 1000 Genomes Project, as will be familiar to the skilled person in the art. In some embodiments, the subject can be classified as being of a particular ancestry if at least the subject's genome comprises a substantial number of different alleles in common with other humans of that ancestry (eg, determined by reference to the 1000 Genomes Project database), for example, at least 10, 20, 30, 40, 50 or 100 or more alleles in common. Abbreviations for particular ancestral groups are provided in

Table 3.

1003231 The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

1003241 Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean + t%.

1003251 As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

1003261 The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in

that description of the embodiment.

1003271 As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

1003281 The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example.' 100329] Definitions of common terms in cell biology and molecular biology can be found in "The Merck Manual of Diagnosis and Therapy", 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al, (eds,), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0- 632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-ID: 0763766321); Kendrew et al, (eds), , Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCI-1 Publishers, Inc., 1995 (ISBN 1-5608L.

569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et a!., eds.

1003301 Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al,, Molecular Cloning: A Laboratory Manual (4 ed), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY., USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A, R, Kimmel Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John B, Coligan, et, al,, ed,, John Wiley and Sons, mc,), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et, al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.

1003311 Other terms are defined herein within the description of the various aspects of the invention, 1003321 All patents and other publications; including literature references, issued patents, published patent applications, arid co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein, These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

1003331 The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate, The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount, These and other changes can be made to the disclosure in light of the detailed description, All such modifications are intended to be included within the scope of the appended claims, 1003341 Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the

disclosure.

1003351 It will be understood that particular configurations, aspects, examples, clauses and embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein, Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of eror for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

1003361 As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps 1003371 Any part of this disclosure may be read in combination with any other part of the disclosure, unless otherwise apparent from the context.

1003381 All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.

While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention, All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[003391 The present invention is described in more detail in the following non limiting

Examples.

[003401 Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs: A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOl), wherein the TOT is present in humans as different polymorphic variants, the method comprising a. selecting a human that is positive for the TOI polymorphic variant, wherein the TOl in said human is encoded by a nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or wherein the TOI in said human is encoded by a nucleotide sequence comprising an allele having total human genotype frequency of less than 50%; and b. administering to the human an anti-TOt ligand to target the TOt in the human to treat or prevent said disease or condition.

2. The method of paragraph 1, wherein before step (a) the ligand has been or is determined as being capable of specifically binding to said TOI variant, 3. The method of paragraph I or 2, comprising determining that the human is positive for the TOl polymorphic variant, optionally wherein the step of determining comprises determining that the human is positive for a nucleotide variant encoding said TOT variant.

4. The method of paragraph 3, wherein the step of determining comprises assaying a biological sample obtained from said human for a nucleotide polymorphism encoding said TOl polymorphic variant, 5. The method of paragraph 3 or 4, wherein the step of determining comprises assaying a biological sample obtained from said human for a protein corresponding to the TOI polymorphic variant.

6. The method of any preceding paragraph, wherein said frequency is less than T5%.

7. The method of any preceding paragraph, wherein said frequency is less than T0%.

8. The method of any preceding paragraph, wherein the ligand is capable of specifically binding to two or more different TOT variants, each being encoded by a nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

9. A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOI), wherein the TOT is present in humans as different polymorphic variants, the method comprising a. selecting a human that is negative for a variant nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; or that the human is negative for a TOT variant encoded by a nucleotide sequence comprising the allele having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; and b. administering to the human an anti-TOT ligand to target the TOT variant in the human and treat or prevent said disease or condition, wherein the TOI in said human is a variant encoded by a nucleotide sequence comprising an allele having a cumulative human allele frequency of more than 50% and/or having a total human genotype frequency of more than 50%.

10. The method of paragraph 9, comprising determining that the human is positive for the TOt polymorphic variant, optionally wherein the determining comprises that the human has been or is phenotyped as positive for the most frequent TOT variant or genotyped for the nucleotide sequence thereof 11 The method of paragraph 10, wherein determining comprises assaying for the nucleotide sequence to determine the presence of said allele.

12. The method of paragraph iT, wherein the assaying comprises nucleic acid amplification.

13. The method of paragraph iT or T2, wherein the assaying comprises hybridization, sequencing, or next generation sequencing.

14. The method of any of paragraphs 11-13, further comprising the step of obtaining a biological sample from the human.

15, The method of any one of paragraphs 9-14, wherein the ligand has been or is determined as being capable of specifically binding to the most frequent TOI variant.

16. The method of any one of paragraphs 9-15, wherein the ligand has been or is determined as being substantially incapable of neutralising or inhibiting said TOT variant.

17. The method of any one of paragraphs 9-16, wherein the ligand is capable of specifically binding to the most frequent TOt variant.

18. The method of any one of paragraphs 9-17, wherein the ligand is capable of specifically binding to two or more different TOT variants, each being encoded by a nucleotide sequence comprising an allele having a cumulative human allele frequency of more than 50%.

19. The method of any preceding paragraph, wherein said TOI polymorphic variant has been or is determined as being present in at least two different human ethnic populations.

20. The method of any preceding paragraph, wherein said cumulative human allele frequency is the frequency in a database of naturally-occurring sequences sampled from at least T5 different human ethnic populations and comprising at least T000 sequences.

2]. The method of any of the preceding paragraphs, wherein the ligand is an antibody, antibody fragment or an affibody.

22. The method of any of the preceding paragraphs, wherein the ligand comprises a nucleotide sequence that specifically hybridises to a TO! nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and!or having a total human genotype frequency of less than 50% or an RNA transcript thereof; and/or the ligand comprises a nucleotide sequence that comprises at least 10 contiguous nucleotides of a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50% or is an anti sense sequence thereof.

23. The method of any of the preceding paragraphs, wherein the genome of said human comprises an allele having a cumulative human allele frequency of less than 50% and the allele is found in at least 2 different ethnic populations.

24. A composition comprising a ligand capable of binding a target of interest encoded by a nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and the allele is found in at least 2 different ethnic populations and optionally a pharmaceutically acceptable caner and optionally a label or instructions for use to treat and/or prevent said disease or condition in a human; optionally wherein the label or instructions comprise a marketing authorisation number issued by a regulatory authority.

25. A kit for treating or preventing a condition or disease mediated by a target of interest as recited in any preceding paragraph, the kit comprising a ligand capable of specifically binding a target of interest encoded by a nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and the allele is found in at least 2 different ethnic populations; and optionally in combination with a label or instructions for use to treat and/or prevent said disease or condition in a human; optionally wherein the label or instructions comprise a marketing authorisation number issued by a regulatory agency; optionally wherein the kit comprises an injection pen or TV container that comprises the ligand.

26. The composition of paragraph 24 or the kit of paragraph 25, wherein the regulatory agency is FDA or EMA.

27. A method of producing an anti-human TOT antibody binding site, the method comprising obtaining a plurality of anti-TOT antibody binding sites, screening the antibody binding sites for binding to a TOT comprising an amino acid sequence encoded by a nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, or to a peptide thereof that comprises an amino acid variation from the corresponding sequence encoded by the TOl-encoding nucleotide sequence comprising an allele having the highest cumulative human allele frequency and/or the highest total human genotype frequency, and isolating an antibody binding site that binds in the screening step.

28. A method of producing an anti-human TO! antibody, the method comprising immunising a non-human vertebrate with a Tot comprising an amino acid sequence encoded by a nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, or to a peptide thereof that comprises an amino acid variation from the corresponding sequence encoded by the TOt-encoding nucleotide sequence comprising an allele having the highest cumulative human allele frequency and/or the highest total human genotype frequency, and isolating an antibody that binds a TOl comprising an amino acid sequence encoded by a TO! nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, and optionally producing a TOI-binding fragment or derivative of the isolated antibody.

29. The method of paragraph 28, wherein the non-human vertebrate is a mouse or a rat.

30. The method of paragraph 29 or 30, comprising the step of obtaining a nucleic acid encoding the antibody, fragment, derivative or binding site and optionally inserting the nucleic acid in an expression vector.

31. A kit for TO! genotyping a human, wherein the kit comprises a nucleic acid comprising a nucleotide sequence that specifically hybridises to a TO! nucleotide sequence selected having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% oranRNAtransciiptthereo and/or the nucleic acid comprises a nucleotide sequence that comprises at least 10 contiguous nucleotides of a TOt nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than SO% or is an antisense sequence thereof.

32. A kit for TO! genotyping or phenotyping a human, wherein the kit comprises a ligand capable of binding a target of interest encoded by a nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% or an antibody, fragment or derivative produced by the method of any one of paragraphs 28 to 30.

33. The kit of paragraph 32, wherein the allele is found in at least 2 different ethnic populations.

34. Use of an anti-TOl ligand that specifically binds a human TO! comprising an amino acid sequence encoded by a TOl nucleotide sequence having a cumulative human allele frequency of less than SO% and/or a total human genotype frequency of less than 50%, in the manufacture of a medicament for treating and/or preventing a TOt-mediated disease or condition in a human whose genome comprises a TO! nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

35. Use of an anti-TOl ligand that specifically binds a human TO! comprising an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, in the manufacture of a medicament for targeting said TOt in a human to treat and/or prevent a disease or condition mediated by TOL 36. A method of targeting a Target of!nterest (TO!) for treating and/or preventing a 101- mediated disease or condition in a human, the method comprising administering an anti-TOt ligand to a human comprising a TOt nucleotide sequence comprising an allele selected as having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, whereby a TO! encoded by said nucleotide sequence is targeted.

37. The method of paragraph 36, wherein the method comprises targeting a human TO! comprising an amino acid sequence with said ligand to treat and/or prevent said disease or condition in said human, wherein said amino acid sequence is encoded by a nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%.

38. A method of Target of Interest (101) genotyping a nucleic acid sample of a human, the method comprising assaying in the sample the presence of a TOt nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

39. A method of Target of Interest (101) typing a protein sample of a human, the method comprising assaying the sample the presence of a TOt amino acid sequence encoded by a TO! nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.

40. The method of paragraph 38 or 39, comprising obtaining a sample of serum, blood, faeces, hair, urine or saliva from a human, whereby the nucleic acid or protein sample is obtained for use in the step of assaying said sequence.

41. The method of any one of paragraphs 38-40, comprising using aligand capable of targeting a nucleic acid sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50% or a ligand capable of specifically binding the TOl encoded by said nucleic acid sequence to carry out said identifying step.

42. A diagnostic kit comprising a ligand that is capable of binding a human Target of Interest (TOT) comprising an amino acid sequence encoded by a TOI nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% and instructions for carrying out the method of any one of paragraphs 38-41 43, The diagnostic kit wherein the ligand is selected from an antibody, antibody fragment, antibody portion, affybody, oligonucleotide, modified oligonucleotide, antisense oligonucleotide, siRNA, and microRNA, 44, A diagnostic kit comprising a nucleic acid probe comprising a nucleotide sequence that specifically hybridises a Target of Interest (TOT) nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or an RNA transcript thereof and instructions for carrying out the method of paragraph 38 or 39.

45. The method, ligand, composition, kit or use of any preceding paragraph, wherein the TOT is encoded by a nucleotide sequence having a cumulative human allele frequency from 1 to 10% and/or a total human genotype frequency from Ito about 15% or from Ito 15%.

46. The method, ligand, composition, kit or use of any preceding paragraph wherein the TOl is a human TOl selected from Table 4; optionally for treating and/or preventing a corresponding disease or condition as set out in Table 4.

47. A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOI), wherein the TOT is present in humans as different polymorphic variants, the method comprising administering to the human determined to be positive for the TOI polymorphic variant, wherein the TOL in said human is encoded by a nucleotide sequence comprising an allele having a cumulative human allele frequency of less than 50% and/or wherein the TOI in said human is encoded by a nucleotide sequence comprising an allele having total human genotype frequency of less than 50% an anti-TOt ligand to target the TOt in the human to treat or prevent said disease or condition.

48. The method of paragraph 47, wherein the anti-TOl ligand is selected from an antibody, an antibody portion, an antibody fragment, an affibody, an antisense oligonucleotide, an siRNA, and a microRNA.

EXAMPLES

1003411 Example 1: Rare PCSK9 Variants 1003421 Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serine protease involved in regulating the levels of the low density lipoprotein receptor (LDLR) protein (Horton et aL, 2007; Seidah and Prat, 2007). In vitro experiments have shown that adding PCSK9 to HepG2 cells lowers the levels of cell surface LDLR (Benjannet et al., 2004; Lagace et al., 2006; Maxwell et al., 2005; Park et aL, 2004). Experiments with mice have shown that increasing PCSK9 protein levels decreases levels of LDLR protein in the liver iBenjannet et al,, 2004; Lagace et al., 2006; Maxwell et al,, 2005; Park et al,, 2004), while PCSK9 knockout mice have increased levels of LDLR in the liver Rashid et al,, 2005).

Additionally, various human PCSK9 mutations that result in either increased or decreased levels of plasma LDL have been identified (Kotowski et al., 2006; Zhao et al., 2006). PCSK9 has been shown to directly interact with the LDLR protein, be endocytosed along with the LDLR, and co-immunofluoresce with the LDLR throughout the endosomal pathway (Lagace et al., 2006).

1003431 PCSK9 is a prohormone-proprotein convertase in the subtilisin (58) family of serine proteases (Seidah et al,, 2003). Humans have nine prohormone-proprotein convertases that can be divided between the S8A and S8B subfamilies (Rawlings et al., 2006). Furin, PC1/PC3, PC2, PACE4, PC4, PC5/PC6 and PC7/PC8/LPC/SPC7 are classified in subfamily SSB. Crystal and NMR structures of different domains from mouse furin and PCI reveal subtilisin-like pro-and catalytic domains, and a P domain directly C-terminal to the catalytic domain (Henrich et al,, 2003; Tangrea et al,, 2002). Based on the amino acid sequence similarity within this subfamily, all seven members are predicted to have similar structures (Henrich et al,, 2005), SKI-I/SIP and PCSK9 are classified in subfamily SSA. Sequence comparisons th these proteins also suggest the presence of subtilisin-like pro-and catalytic domains (Sakai et al., 1998; Seidah et al., 2003; Seidah et al., 1999). In these proteins the amino acid sequence C-terminal to the catalytic domain is more variable and does not suggest the presence of a P domain.

1003441 Prohormone-proprotein convertases are expressed as zymogens and they mature through a multi step process. The function of the pro-domain in this process is two-fold. The pro-domain first acts as a chaperone arid is required for proper folding of the catalytic domain (Ikemura et a!,, 1987). Once the catalytic domain is folded, autocatalysis occurs between the pro-domain and catalytic domain. Following this initial cleavage reaction, the pro-domain remains bound to the catalytic domain where it then acts as an inhibitor of catalytic activity (Fu et al., 2000). When conditions are correct, maturation proceeds with a second autocatalytic event at a site within the pro-domain (Anderson et al,, 1997). After this second cleavage event occurs the pro-domain and catalytic domain dissociate, giving rise to an active protease.

1003451 Autocatalysis of the PCSK9 zymogen occurs between G1n152 and SeriS3 (VEAQIS I P) (Naureckiene et al,, 2003), and has been shown to be required for its secretion from cells (Seidah et al,, 2003), A second autocatalytic event at a site within PCSK9's pro-domain has not been observed. Purified PCSK9 is made up of two species that can be separated by non-reducing SDS-PAGE; the pro-domain at 17 Kd, and the catalytic plus C-terminal domains at 65 Kd. PCSK9 has not been isolated without its inhibitory pro-domain, and measurements of PCSK9's catalytic activity have been variable (Naureckiene et al., 2003; Seidahetal,,2003), 1003461 lii certain embodiments, a PCSK9 polypeptide includes terminal residues, such as, but not limited to, leader sequence residues, targeting residues, amino temiinal methionine residues, lysine residues, tag residues and/or frision protein residues. "PCSK9" has also been referred to as FH3, NARC1, HCHOLA3, proprotein convertase subtilisin/kexin type 9, and neural apoptosis regulated convertase 1. The PCSK9 gene encodes a proprotein convertase protein that belongs to the proteinase K subfamily of the secretory subtilase family. The term "PCSK9" denotes both the proprotein and the product generated following autocatalysis of the proprotein. When only the autocatalyzed product is being referred to (such as for an antigen binding protein or ligand that binds to the cleaved PCSK9), the protein can be referred to as the "mature," "cleaved", "processed" or "active" PCSK9. When only the inactive form is being referred to, the protein can be referred to as the "inactive", "pro-form", or "unprocessed" form of PCSK9, The term PCSK9 also encompasses PCSK9 molecules incorporating post-translational modifications of the PCSK9 amino acid sequence, such as PCSK9 sequences that have been glycosylated, PCSK9 sequences from which its signal sequence has been cleaved, PCSK9 sequence from which its pro domain has been cleaved from the catalytic domain but not separated from the catalytic domain (see, e.g., FIGS. IA and lB ofUS2O120093818A1; which is incorporated by reference herein in its entirety).

[003471 The present invention provides affli-PCSK9 ligands; and PCSK9-binding or targeting ligands as described herein. The ligands have a variety of utilities. Some of the ligands, for instance, are useful in specific binding assays, for genotyping or phenotyping humans, affinity purification of PCSK9, in particular human PCSK9 or its ligands and in screening assays to identify other antagonists of PCSK9 activity. Some of the ligands of the invention are useful for inhibiting binding of PCSK9 to LDLR, or inhibiting PCSK9-mediated activities.

[003481 Anti-PCSK9 ligands (eg, antibodies and anti-sense RNA) have been developed based on targeting and neutralising so-called "wild-type" human PCSK9, which is a commonly-occurring form (see, eg, US20I200938]8AI and US2OI I0065902AI; each of which is incorporated by reference herein in its entirety). While such therapies are useful for human patients harbouring this form of human PCSK9, the inventor considered it useffil to investigate the possibility of targeting much rarer -but still naturally-occurring -forms of PCSK9 amongst human populations, k this way, the inventor arrived at insight into the natural occurrences and distributions of rarer human PCSK9 forms that can serve as useftil targets (at the protein or nucleic acid level) for human treatment, prophylaxis md diagnosis pertinent to diseases and conditions mediated or associated with PCSK9 activity. This particularly provides for tailored therapies, prophylaxis and diagnosis in humans that are devoid of the common PCSK9 gene or protein (ie, the form a or ci' as used in US201200938 I 8A1 and TJS2O1 1 0065902A1 to generate antibodies), [003491 The skilled person will know that SNPs or other changes that translate into amino acid variation can cause variability in activity and/or conformation of human targets to be addressed. This has spawned great interest in personalized medicine where genotyping and knowledge of protein and nucleotide variability is used to more effectively tailor medicines and diagnosis of patients. The invention, therefore, provides for tailored pharmaceuticals and testing that specifically addresses rarer PCSK9 polymorphic variant forms. Such forms or "alleles" (at the nucleotide level), in many of the examples determined by the inventor, comprise multiple changes at the nucleotide and amino acid levels from the corresponding common form nucleotide and amino acids sequences, ie, there are multiple non-synonymous changes at the nucleotide level that translate into multiple corresponding changes in the protein target in humans.

1003501 Furthermore, the inventor surprisingly realised that the rarer natural forms, although present in humans at much lower frequencies than the common form, nevertheless are represented in multiple and ethnically-diverse human populations and usually with many human examples per represented ethnic population. Thus, the inventor realised that targeting such rarer forms would provide for effective treatment, prophylaxis or diagnosis across many human ethnic populations, thereby extending the utility of the present invention.

1003511 With this realisation, the inventor realised that there is significant industrial and medical application for the invention in terms of guiding the choice of anti -PCSK9 ligand for administration to human patients for therapy and/or prophylaxis of PCSK9-mediated or associated diseases or conditions. In this way, the patient receives drugs and ligands that are tailored to their needs -as determined by the patient's genetic or phenotypic makeup. Hand-in-hand with this, the invention provides for the genotyping and/or phenotyping of patients in connection with such treatment, thereby allowing a proper match of drug to patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment using drugs or ligands that are not matched to the patient (eg, poor efficacy and/or side-effects) and avoids pharmaceutical mis-prescription and waste.

1003521 In developing this thinking, the present inventor decided to determine a set of human PCSK9 variants on the basis of the following criteria, these being criteria that the inventor realised would provide for useful medical drugs and diagnostics to tailored need in the human population. The inventor selected variants having at least 3 of the 4 following criteria:- * PCSK9 variants having a cumulative human allele frequency in the range from 1 to 10%; * PCSK9 variants having a total human genotype frequency in the range from t to about 15%; * PCSK9 variants found in many different human ethnic populations (using the standard categorisation of the 1000 Genomes Project, which is an accepted standard in the art; see Table 3 below); and * PCSK9 variants found in many individuals distributed across such many different ethnic populations.

1003531 On the basis of these criteria, the inventor identified the variants listed in Table 1 below (excluding form a).

[00354J The inventor's selection included, as a consideration, selection for nucleotide variation that produced amino acid variation in corresponding PCSK9 fonns (ie, non-synonymous variations), as opposed to silent variations that do not alter amino acid residues in the target protein.

Table 1: Human PCSK9 variants distributed over several human ethnic populations & having a total human genotype frequency in the range of ito about 15% (a) Amino acid variability, population distributions and frequencies Form a 46R 53A 425N 443A 4741 619Q 6/CE ASW,YRI,GBR, 939 14 0.3951 4506 064815 T5I,CLM,CI-1B, (O8457) LWK,Cl-I$1MXL, JPT PLJR,IBS,FIN,

______ ____ ____ _____ _____ _____ _____ _____ C EU _________ _________ ________ ____________ ________

Variant _____ Amino Acid Position & Variation ______ Human No. No. Unique Het Freq3 Hom Freq4 Cum Freq6 Form 46L 53V 4255 443T 474V 6191' 670G Populations Individs1 Pops2 (Het + Horn ________ _____ _____ ______ ______ ______ ______ ______ ________________ ___________ ___________ __________ freq5) ___________ f x ASW,YRI,GBR, 180 12 0.153 0.009 0.0855 TSI,CLM,LWK,M (0.162) XL,JPT,PUR,IBS, _______ _____ _____ _____ _____ _____ _____ _____ FIN,CEU __________ __________ _________ _____________ _________ c x ASW,YRI,GBR, 153 12 0.1296 0.0081 0.0729 TSI,CLM,CHB, (0.1377) LWK,CHS,JPT, ________ _____ _____ ______ ______ ______ ______ ______ PUR,FIN,CEU ___________ ___________ _________ ______________ __________ r x x 0.0234 0.009 0.0292 ________ _____ _____ ______ ______ ______ ______ ______ ________________ ___________ ___________ __________ (0.0324) ___________ p x x ASW,GBR,T5l, 49 9 0.0441 (0.0441) 0.0221 CLM,JPT,PUR, ________ _____ _____ ______ ______ ______ ______ ______ IBS,FIN,CEU ___________ ___________ __________ _______________ ___________ m x LWK,ASW,YRI, 29 4 0.0225 (0.0225) 0.0149

_____ ____ ____ ____ ____ ____ ____ ____ CLM ________ ________ _______ __________ _______

e _____ _____ x ______ x ______ ______ LWK,ASW,YRI 15 3 0.0135 (0.0135) 0.0068 h _____ _____ ______ x ______ x ______ LWK,ASW,VRI 10 3 0.009 (0.009) 0.0045 aj X x ______ ______ PUR,TSI,FIN,CEU 9 4 0.0081 (0.0081) 0.0041 q x x CHS,ASW,JPT, 7 5 0.0063 (0.0063) 0.0032 _______ _____ _____ _____ _____ _____ _____ _____ PUR,CHB __________ __________ _________ _____________ _________

Table Footnotes:

"x" in a box indicates that the amino acid for the variant form is different from the amino acid at that position in form a, the variant amino acid being shown in Amino Acid Position & Variation" of the table and the form a amino acid being shown in the first row of the table; amino acids at all other positions of each variant fonn are identical to those found in form a.

Amino acid numbering is per the numbering shown for the pro-form in Table 2 below.

1. Number of individuals in 1000 Genomes database found to have the allele; 2. Number of unique human ethnic populations in 1000 Genomes database in which the allele was found to occur; 3. lileterozygous human genotype frequency, ie, cumulative frequency of all genotypes having one occurrence of the variant allele and one occurrence of another allele (heterozygous state), eg, ac genotype in 1000 Genomes database; 4. Homozygous human genotype frequency, ie, cumulative frequency ot two occurrences of the vanant allele (homozygous state), eg, cc a genotype in 1000 Genomes database; and 5. Total human genotype frequency, ie, total of heterozygous plus homozygous human genotype frequencies.

6. Cumulative human allele frequency of all occurrences of the variant allele in 1000 Genomes database.

Form a' is identical to form a with the exception that form a' has a glycine (G) at position 620 (see (JS20 120093818 (Amgen, mc)); form a has E at this position (b) Nucleotide Sequence Variations of Selected Alleles Alielea I C A A I A A _____________ Nucleotide_Position1 Variant Allele 1:55505647 1:55505668 1:55523802 1:55523855 1:55524237 1:55527222 1:55529187 Non-Synonymous Nucleoticle VarItian2 iji G IA] C I C G Variant lD rs11591147 rs11583680 rs28362261 rs28362263 rs562556 rs28362277 rs5OSlSl ____________ ____________ Corresponding Amino Acid Variation ____________ ____________ _____________ 46L 53V 425S 4431 474V 619P 670G f _____ _____ _____ _____ X _____ _____ c X r ______ ______ ______ ______ X ______ X -.4 w p _____ x x m _____ _____ _____ X _____ _____ _____ e X X h _____ _____ _____ X _____ X _____ aj X ____________ ____________ ____________ X ____________ ____________ q _____ X _____ _____ _____ _____ X "x'1 in a box indicates that a variant allele comprises the non-synonymous nucleotide variation indicated in the 5th row.

Table Footnotes:

1. Notation is chromosome number (all positions are on human chromosome i):coordinate number (Ensembl release 73 -September 2013, Genome assembly: GRCh37 (GCA_00000 1405.13); 2. Nucleotide change (compared to allele a nucleotide shown in first row) giving rise to an amino acid change in the variant form (compared to amino acid of allele a); and 3. NCBI dIISNP reference number (NCBI dbSNP Build 138 released on Apr 25, 2013).

Table 2: Sequences

(a) Human PCSKB Form a Amino Acid Sequence (SEQ ID NO:1) -"Pro-form" with Signal Sequence 1-5 53

MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLAL-RSEEDGLAEAPEHGTTATFHRCAKDP

WPLPGTYJVLKEETHLSQSERTPLRRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPH

VDY I -4 5

425 443 474 dvneawfpedqMtpnvaapps:thGAGWQLFCRTVWSAHSGPTRM.AT. MARCAPDEELSCSSfSRSGKRRGERM 0) EAQGGKVCR.AHNAFGGEGVYAARCCU9QANCSVHTAPPAEASMGTRVHc.HQQGHVLTGCSSHWEVED[GT 519 520 HKPPV1RPRGQPNQCVGHREAS HASCCHAPGftCKVKEHG PAPQVTVACEEGW1LTGcSAIP6TSHVLGAY AVDNTCVVRSRDVS1ITGSTSEEAVTAVNCCRSRHL4QASQEIQ Italics = signal sequence 1-3D Courier = pro peptide 31-152 lower case = catalytic domain 153-449 UPPER CASE = C-terminal domain 450-692 Underlined = residues changed from allele a in other sequences (aa residue number shown) The pro-form is the sequence from amino acid number 3 1 to (and including) amino acid number 692 of SEQ ID NO: 1.

The mature form is the sequence from amino acid number 153 to (and including) amino acid number 692 of SEQ ID NO: 1.

(b) iltiman PCSK9 Form a Amino Acid Sequence (SEQ ID NO:3) -"Mature-form" (Numbering and notation as per SEQ ID NO:1 above has been retained) spwn.lerftppryradeyqppdggskevylldtsiq: sdhceiegrvmvtdfenvpeedgtrfhrqaskcdshg 425 443 474 thnea fpedqMtpnIvaappsthGAGWQLFCRWW.SAHSGPTRMA1AiARCAPDEELLSCSSFSRSGKRRGERM EAQGKLVCRAHNAF&GEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTCSSHWEVEEWGT HKPPVLRPRGQPNQCVG HREAS1HASCCHAPGLECKYKEHGPAPQQvTVACEEGWTLTGCSALPGTSHVLGAY AVDNTCVVRSRDVSTTGSTSEEAVTAVMCcFSRHLAQASQELQ (c) Human PCSKO Allele a Nucleotide Sequence (SEQ ID NO: 28)-Encoding "Pro-form" Plus Signal Sequence 4TGGGCACCGTCAGCTCCAGGCGGTCCT*GGTGGCCGCTGCC4 CTGCTGCTGCTGCTGCTGCTG CTCCTGGGTC R4ÔLCGTIo CU c*ccc A53V&CCWC-TC

A

CCCTGCACGT

CACTGCCCGCCGCCTGCKGGCCCAGrTG.CCCGCCGGG.GATACCTCACCAAGKTCCTG.CATGTCTCC

ATCC-CCTTCT T Co

C

atcgagggcagggtcatggtc.accgacttcgagaatgtgcccgaggaggacgggacccgcttc. cacagacaggcaag.caagtgtgacagtc;t a g g g gacatc.acaggctgctgcccac. gtggctggcttgcagccatgatgctgtctgccgagccggagctcaacctggccg:agttg.aggcagagactg N4Z5SAATIoWT A443TGCCtoAU atc:cacttcctg:ccaaag.atgtcatcaatga ggcctggttccctgaggaccagcgggtactgac:ccccaa:cctggtggccgççctgccccccag cacccatGCGGCAGGTTGGCAGCTGTITIGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC 1474.V ATC.to ETC ACAGCCATCGCCCCTGCGCCCCAGPTGAGGAcCTGCTGAGCTG:CTCCAGTTTCTCCAGGAGTGGAAGCG

C

GTGTCTACGCCATTGCCA: GGIGCTGCCTGCTACCCCAGGGCMCTGCAGCGTCCACACAGCTCCACCAGCTGA GGCC.AGC.ATGGGGACCCGTGTCCACIGCCACC. AACAGG*GCCACGTCCICACAG*GCTGCAGCTCCCACTGGGA GGTGGAGGACCHGGCAC*CCACAAGCC*G*CCTGT*G*CTGAGGCCACGAGG1CAGCCCMCCAGTGCGTGGGCC ACAGG*GAGGCCAGCATCCAC*GCTft.CTGCTGCC.ATGCCCCAGGTCTGGAATGCMAGTCMGGAGCMGGAA Q&19PCAG t ccc EGZOG GAG teGGG

T ______

CTGGACCTCCCACGTCCTGGGGGCCTACGCCETAGACAACACGTGTGTAGTCAGGAGCCGGACGTCA:GCA E7%. GAG WGG3 CTACACAGCACCAGCGAN3AG3CCGTGACAGCC.TTGCCATCTGCTCC:GAGCCG:CACCJGGCGCAG GC.CTCCCAGGAGCTC.CAGTGAC italics = nucleotide sequence encoding signal sequence (nucleotides 1-90) Courier = nucleotide sequence encoding pro peptide (nucleotides 91-456) lower case = nucleotide sequence encoding catalytic domain (nucleotides 457-1346) LTPPER CASE = nucleotide sequence encoding C-terminal domain (nucleotides 1347-2076) Underlined = allelic variations from allele a in other sequences (aa residue number changes and codon changes shown) The pro-fonn is encoded by nucleotide sequence from nucleotide 91 to (and including) nucleotide 2076.

The mature form is encoded by nucleotide sequence from nucleotide 457 to (and including) nucleotide 2076.

C 0:,

C

[003551 Variant Allele Nucleotide Sequences Thus, (i) The nucleotide sequence of allele f is identical to SEQ ID NO: 28 except that the nucleotide sequence of allelef comprises a GTC codon instead of an ATC codon at the position labelled "1474V" in SEQ ID NO: 28; (ii) The nucleotide sequence of allele c is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele c comprises a GGG codon instead of an GAG codon at the position labelled "E670G" in SEQ ID NO: 28; (iii) The nucleotide sequence of allele r is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele r comprises a GTC codon instead of an ATC codon at the position labelled "1474V" in SEQ ID NO: 28; and a GGG codon instead of an GAG codon at the position labelled "EÔ7OG" in SEQ ID NO: 28; (iv) The nucleotide sequence of allelep is identical to SEQ ID NO: 28 except that the nucleotide sequence of allelep comprises a GTC codon instead of a 0CC codon at the position labelled "AS3V' in SEQ ID NO: 28; and a GTC codon instead of an ATC codon at the position labelled "1474V" in SEQ ID NO: 28; (v) The nucleotide sequence of allele iii is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele m comprises a ACC codon instead of a 0CC codon at the position labelled "A443T" in SEQ ID NO: 28; (vi) The nucleotide sequence of allele e is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele e comprises a AGT codon instead of an AAT codon at the position labelled "N4255" in SEQ ID NO: 28; and a GTC codon instead of an ATC codon at the position labelled "1474V" in SEQ ID NO: 28; (vii) The nucleotide sequence of allele his identical to SEQ ID NO: 28 except that the nucleotide sequence of allele/i comprises a ACC codon instead of a GCC codon at the position labelled "A443T" in SEQ ID NO: 28; and a CCG codon instead of a CAG codon at the position labelled "QoI9P" in SEQ ID NO: 28; (viii) The nucleotide sequence of allele a/is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele cij comprises a CTT codon instead of an COT codon at the position labelled "R46L" in SEQ ID NO: 28; and a GTC codon instead of an ATC codon at the position labelled 1474V" in SEQ ID NO: 28; and (ix) The nucleotide sequence of allele q is identical to SEQ ID NO: 28 except that the nucleotide sequence of allele q comprises a GTC codon instead of a 0CC codon at the position labelled "A53V" in SEQ ID NO: 28; and a GGG codon instead of an GAG codon at the position labelled "E6700" in SEQ ID NO: 28.

[003561 Variant Pro-Form Amino Acid Sequences (Numbering is as per SEQ lID NO: I recited above) (A) The amino acid sequence of form/is identical to the amino acid sequence from amino acid number 3 to (and including) amino acid number 692 of SEQ ID NO: I except that the amino acid sequence of formfcomprises a valine at position 474; (B) The amino acid sequence of form c is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: except that the amino acid sequence of form c comprises a gycine at position 670; (C) The amino acid sequence of form r is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: except that the amino acid sequence of form r comprises a valine at position 474 and a glycine at position 670; (D) The amino acid sequence offormp is identical the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: I except that the amino acid sequence of form p comprises a valine at position 53 and a valine at position 474; (E) The amino acid sequence of form in is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form in comprises a threonine at position 443; (F) The amino acid sequence of form e is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form e comprises a serine at position 425 and a valine at position 474; (G) The amino acid sequence of form his identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form h comprises a threonine at position 443 and a proline at position 619; (H) The amino acid sequence of form q,/ is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form aj comprises a leucine at position 46 and a valine at position 474; and (I) The amino acid sequence of form q is identical to the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1 except that the amino acid sequence of form q comprises a valine at position 53 and a glycine at position 670.

[003571 Variant Mature Form Amino Acid Sequences (Numbering is as per SEQ lID NO: I recited above) (A') The amino acid sequence of form/is identical to SEQ ID NO: 2 except that the amino acid sequence of form fcomprises a valine at position 474; (B') The amino acid sequence of form c is identical to SEQ ID NO: 2 except that the amino acid sequence of form c comprises a glycine at position 670; (C') The amino acid sequence of form r is identical to SEQ ID NO: 2 except that the amino acid sequence of form r comprises a valine at position 474 and a glycine at position 670; (D') The amino acid sequence offormp is identical to SEQ ID NO: 2 except that the amino acid sequence of form p comprises a valine at position 474; (E') The amino acid sequence of form in is identical to SEQ ID NO: 2 except that the amino acid sequence of form in comprises a threonine at position 443; (F') The amino acid sequence of form e is identical to SEQ ID NO: 2 except that the amino acid sequence of form e comprises a serine at position 425 and a valine at position 474; (C) The amino acid sequence of form his identical to SEQ ID NO: 2 except that the amino acid sequence of form h comprises a threonine at position 443 and a proline at position 619; (H') The amino acid sequence of form aj is identical to SEQ ID NO: 2 except that the amino acid sequence of form aJ comprises valine at position 474; and (I') The amino acid sequence of form q is identical to SEQ ID NO: 2 except that the amino acid sequence of form q comprises a gycine at position 670.

[003581 The mature form ofp is identical to the mature form of /and cii.

[003591 The mature form of c is identical to the mature form of q.

[003601 Further sequence analysis and 3D En silica modelling (see Figure 1) revealed that selected variants also frilfilled the foflowing selection criteria:- * PCSK9 variants whose variant amino acid residues (versus the common form of human PCSK9) are found in the mature form of the target (ie, outside the pro-domain); and * PCSK9 variants whose variant amino acid residues (versus the common form of human PCSK9) are surface-exposed on the target, which the inventor saw as contributing to determining the topography of the target and potentially contributing to how and where ligand binding on the target occurs.

1003611 As shown in Figure 1, identified positions 425, 443, 474, 619 and 670 (found in the selected variants of the invention) are all surface-exposed and outside of the pro-domain.

Variant positions 425 and 443 are surface-exposed on the catalytic domain, while variant positions 474, 619 and 670 are surface-exposed on the C-terminal domain.

1003621 In a first example, the invention addresses the need to treat humans having naturally-occurring rarer natural PCSK9 alleles, genotypes and phenotypes (rarer protein forms). In this respect, the invention provides the following aspects: [003631 In a First Aspect: An anti-human PC 51(9 ligand for use in a method of treating and/or preventing a PCSK9-mediated disease or condition in a human whose genome comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, wherein the method comprises administering the ligand to the human.

[003641 lii an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the oup consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37.

These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

[003651 In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta c/ a! 2006).

[003661 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 6700 which is a marker for severity of coronary atherosclerosis (Chen et at 2005).

[003671 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32,34,35,36 and 37; or selected from the group consisting of SEQ ID NOs: 3], 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

[003681 In an example, the nucleotide sequence is SEQ ID NO: 29.

[003691 In an example, the nucleotide sequence is SEQ ID NO: 30.

[003701 In an example, the nucleotide sequence is SEQ ID NO: 31.

1003711 In an example, the nucleotide sequence is SEQ ID NO: 32.

1003721 In an example, the nudeotide sequence is SEQ ID NO: 33.

[003731 In an example, the nucleotide sequence is SEQ ID NO: 34.

[003741 In an example, the nucleotide sequence is SEQ ID NO: 35.

[003751 In an example, the nucleotide sequence is SEQ ID NO: 36.

[003761 In an example, the nucleotide sequence is SEQ ID NO: 37.

[003771 In a Second Aspect: The ligand of aspect 1, wherein the ligand has been or is determined as capable of binding a human PCSK9 selected from the group consisting forms f C, i p, in, e, h, cif and q.

[003781 In an example of any aspect, the ligand binds (or has been determined to bind) two, three, four or more human PCSK9 selected from the group consisting forms / c, r, p, in, e, h, cej and q.

[003791 In an example of any aspect, the ligand comprises a protein domain that specifically binds to PCSK9, eg, a human PCSK9 selected from the group consisting formsf C, F, p, in, e, h, aj and q.

[003801 The term "specifically binds," or the like, means that a ligand, eg, an antibody or antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about i>< t06 M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like, An isolated antibody that specifically binds a human PCSK9 may, however, exhibit cross-reactivity to other antigens such as a PCSK9 molecule from another specie. Moreover, multi-specific antibodies (e.g., bispecifics) that bind to human PCSK9 and one or more additional antigens are nonetheless considered antibodies that "specifically bind" PCSK9, as used herein.

[003811 In an example of any aspect, the ligand comprises or consists of a protein that mimics the EGFA domain of the LDL receptor and specifically binds to PCSK9, eg, a human PCSK9 selected from the group consisting formsf c, r, p, in, e, h, a/and q.

[003821 In an example of any aspect, the ligand antagonises PCSK9, eg, a human PCSK9 selected from the group consisting formsf c, r, p, in, e, h, af and q.

[003831 In an example of any aspect, the method comprises (before administering the ligand) the step of determining that the ligand is capable of binding a human PCSK9 selected from the group consisting formsj c, r, p, in, e, /i, cxi and q.

1003841 In an example of any aspect, binding is determined by SPR, In an example of any aspect, binding is determined by ELISA.

[003851 In an example of any aspect, said forms are the mature forms.

[003861 In an example of any aspect, said forms are the pro-forms.

[003871 In a Third Aspect: A ligand that binds a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27 for use in a method comprising the step of using the ligand to target said PCSK9 in a human to treat and/or prevent a disease or condition mediated by PCSK9, the method comprising administering the ligand to the human.

[003881 In an example, the disease or condition is mediated by a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27.

[003891 In an example, the amino acid sequence selected from the group consisting of SEQ ID NOs: 4-23, 26 and 27; or selected from the group consisting of SEQ ID NOs: 4-14 and 18-27; or selected from the group consisting of SEQ ID NOs: 4-14, t8-23, 26 and 27.

These are naturally-occurring sequences that do not comprise 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

[003901 In an example, the amino acid sequence is SEQ ID NO: 18, 190120, that comprises a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

[003911 In an example, the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 26 and 27, that comprise 6706 which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

[003921 In an example, the amino acid sequence selected from the group consisting of SEQ ID NOs: 10-14 and 18-27; or selected from the group consisting of SEQ ID NOs: 10-14, 18-23, 26 and 27. These are sequences that have a naturally-occurring combination of differences from SEQ ID NOs: 1-3 (form a) and which meet the criteria set out above.

[003931 In an example, the amino acid sequence is SEQ ID NO: 4.

[003941 In an example, the amino acid sequence is SEQ ID NO: 5.

[003951 In an example, the amino acid sequence is SEQ ID NO: 6.

[003961 In an example, the amino acid sequence is SEQ ID NO: 7.

[003971 In an example, the amino acid sequence is SEQ ID NO: 8.

[003981 In an example, the amino acid sequence is SEQ ID NO: 9.

[003991 In an example, the amino acid sequence is SEQ ID NO: 10.

[004001 In an example, the amino acid sequence is SEQ ID NO: 11.

[004011 In an example, the amino acid sequence is SEQ ID NO: 12.

1004021 In an example, the amino acid sequence is SEQ ID NO: 13.

1004031 In an example, the amino acid sequence is SEQ ID NO: 14.

[004041 In an example, the amino acid sequence is SEQ ID NO: 15.

[004051 In an example, the amino acid sequence is SEQ ID NO: 16.

[004061 In an example, the amino acid sequence is SEQ ID NO: 17.

[004071 In an example, the amino acid sequence is SEQ ID NO: 18.

[004081 In an example, the amino acid sequence is SEQ ID NO: 19.

[004091 In an example, the amino acid sequence is SEQ ID NO: 20.

[004101 In an example, the amino acid sequence is SEQ ID NO: 21.

[004111 In an example, the amino acid sequence is SEQ ID NO: 22.

[004121 In an example, the amino acid sequence is SEQ ID NO: 23, [004131 In an example, the amino acid sequence is SEQ ID NO: 24.

[004141 In an example, the amino acid sequence is SEQ ID NO: 25.

[004151 In an example, the amino acid sequence is SEQ ID NO: 26.

[004161 In an example, the amino acid sequence is SEQ ID NO: 27.

[004171 In a Fourth Aspect: The ligand of aspect 3, wherein the genome of the human comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37.

[004181 In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the oup consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37.

[004191 In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et a! 2006).

[004201 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et at 2005).

[004211 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

[004221 In an example, the nucleotide sequence is SEQ ID NO: 29 1004231 In an example, the nucleotide sequence is SEQ ID NO: 30.

1004241 In an example, the nucleotide sequence is SEQ ID NO: 31.

[004251 In all example, the nucleotide sequence is SEQ ID NO: 32.

[004261 In an example, the nucleotide sequence is SEQ ID NO: 33 [004271 In an example, the nucleotide sequence is SEQ ID NO: 34 [004281 In an example, the nucleotide sequence is SEQ ID NO: 35, [004291 In an example, the nucleotide sequence is SEQ ID NO: 36.

[004301 In an example, the nucleotide sequence is SEQ ID NO: 37.

[004311 In a Fifth Aspect: The ligand of any preceding aspect, wherein the human has been or is genotyped as positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-3 7 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof [004321 In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37.

[004331 In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta eta! 2006).

[004341 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen ci at 2005).

[004351 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

[004361 In an example, the nucleotide sequence is SEQ ID NO: 29.

[004371 In an example, the nucleotide sequence is SEQ ID NO: 30.

[004381 In an example, the nucleotide sequence is SEQ ID NO: 3].

[004391 In an example, the nucleotide sequence is SEQ ID NO: 32.

[004401 In an example, the nucleotide sequence is SEQ ID NO: 33.

[004411 In an example, the nucleotide sequence is SEQ ID NO: 34.

1004421 In an example, the nucleotide sequence is SEQ ID NO: 35, 1004431 In an example, the nucleotide sequence is SEQ ID NO: 36.

[004441 In an example, the nucleotide sequence is SEQ ID NO: 37.

[004451 In a Sixth Aspect: The ligand of any preceding aspect, wherein the human has been or is phenotyped as positive for a human PCSK9 selected from the group consisting of forms f c, ; p, m, e, h, aj and q or at least the catalytic or C-terminal domain thereof [004461 In an example, said forms are the mature forms.

[004471 In an example, said forms are the pro-forms.

[004481 In a Seventh Aspect: The ligand of any preceding aspect, wherein the method comprises genotyping the human as positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-3 7 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof [004491 In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37.

These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above, These groups comprise variants that are associated with elevated LDL-C.

[004501 In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta eta! 2006).

[004511 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen ci at 2005).

[004521 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

[004531 In an example, the nucleotide sequence is SEQ ID NO: 29.

[004541 In an example, the nucleotide sequence is SEQ ID NO: 30.

[004551 In an example, the nucleotide sequence is SEQ ID NO: 3].

[004561 In an example, the nucleotide sequence is SEQ ID NO: 32.

[004571 In an example, the nucleotide sequence is SEQ ID NO: 33.

[004581 In an example, the nucleotide sequence is SEQ ID NO: 34.

1004591 In an example, the nucleotide sequence is SEQ ID NO: 35, 1004601 In an example, the nucleotide sequence is SEQ ID NO: 36.

[004611 In an example, the nucleotide sequence is SEQ ID NO: 37.

[004621 In an Eighth Aspect: The ligand of any preceding aspect, wherein the method comprises phenotyping the human has positive for a human PCSK9 selected from the group consisting of forms f c, r, p. m, e, h, csj and q or at least the catalytic or C-terminal domain thereof.

[004631 In an example, said forms are the mature forms.

[004641 In an example, said forms are the pro-forms.

[004651 In a Ninth Aspect: The ligand of any preceding aspect, wherein the human has been or is genotyped as heterozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof optionally wherein the human has been or is genotyped as comprising the nucleotide sequence of SEQ ID NO: 28 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-3 7 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof [004661 "Heterozygous" here means that in the human's genotype one allele comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof and other allele can be any PCSK9 (eg, form a, a' or an allele comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-3 7 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof).

[004671 In an example, the method comprises (before administering the ligand) genotyping the human as heterozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof optionally also genotyping the human as comprising the nucleotide sequence of SEQ ID NO: 28 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof [004681 In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37.

1004691 These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

1004701 In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciofta el ci 2006).

1004711 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et ci 2005), 1004721 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

1004731 In an example, the nucleotide sequence is SEQ ID NO: 29.

1004741 In an example, the nucleotide sequence is SEQ ID NO: 30.

1004751 In an example, the nucleotide sequence is SEQ ID NO: 31.

1004761 In an example, the nucleotide sequence is SEQ ID NO: 32.

1004771 In an example, the nucleotide sequence is SEQ ID NO: 33.

1004781 In an example, the nucleotide sequence is SEQ ID NO: 34.

1004791 In an example, the nucleotide sequence is SEQ ID NO: 35.

1004801 In an example, the nucleotide sequence is SEQ ID NO: 36.

1004811 In an example, the nucleotide sequence is SEQ ID NO: 37.

1004821 In a Tenth Aspect: The ligand of any one of aspects 1 to 9, wherein the genome of the human has been or is genotyped as homozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-3 7 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof 1004831 "Homozygous" here means that in the human's genotype each allele comprises the same nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-3 7 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof 1004841 In an example, the method comprises genotyping the human as homozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof 1004851 In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the woup consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37.

1004861 In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et a/ 2006).

1004871 In an example, the nudeotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen etaI200S).

1004881 In an example, the nudeotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37, These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

1004891 In an example, the nucleotide sequence is SEQ ID NO: 29.

1004901 In an example, the nucleotide sequence is SEQ ID NO: 30.

1004911 In an example, the nucleotide sequence is SEQ ID NO: 3].

1004921 In an example, the nucleotide sequence is SEQ ID NO: 32.

1004931 In an example, the nucleotide sequence is SEQ ID NO: 33.

1004941 In an example, the nucleotide sequence is SEQ ID NO: 34.

1004951 In an example, the nudeotide sequence is SEQ ID NO: 35.

1004961 In an example, the nucleotide sequence is SEQ ID NO: 36.

1004971 In an example, the nucleotide sequence is SEQ ID NO: 37.

1004981 In an Eleventh Aspect: The ligand of any preceding aspect, wherein the ligand comprises an antibody binding site that binds a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27 and optionally has been or is determined as capable of such binding.

1004991 In an example, the method comprises (before administering the ligand) the step of determining that the ligand is capable of binding to said human PC SK9.

1005001 In an example, the binding is specific binding. In an example, the ligand binds (or has been determined as binding) to the PCSK9 with an affinity (Kd) of] mM, I OOnM, I OnM or lnlVI or less. In an embodiment, the affinity is no less than 10, 100 or 1000 fivI.

1005011 In an example, binding or affinity is determined by SPR or ELISA.

1005021 In an example, the disease or condition is mediated by a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27.

[005031 In all example, the amino acid sequence selected from the group consisting of SEQ ID NOs: 4-23, 26 and 27; or selected from the group consisting of SEQ ID NOs: 4-14 and 18-27; or selected from the group consisting of SEQ ID NOs: 4-14, 18-23, 26 and 27.

[005041 In an example, the amino acid sequence is SEQ ID NO: 18, 19 0120, that comprises a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

[005051 In an example, the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 26 and 27, that comprise 6700 which is a marker for severity of coronary atherosclerosis (Chen et al 2005).

[005061 In an example, the amino acid sequence selected from the group consisting of SEQ ID NOs: 10-14 and 18-27; or selected from the group consisting of SEQ ID NOs: 10-14, 18-23, 26 and 27. These are sequences that have a naturally-occurring combination of differences from SEQ ID NOs: 1-3 (fonn a) and which meet the criteria set out above [005071 In an example, the amino acid sequence is SEQ ID NO: 4.

[005081 hI an example, the amino acid sequence is SEQ ID NO: 5.

[005091 hI an example, the amino acid sequence is SEQ ID NO: 6.

[005101 In an example, the amino acid sequence is SEQ ID NO: 7.

[005111 In an example, the amino acid sequence is SEQ ID NO: 8.

[005121 hI an example, the amino acid sequence is SEQ ID NO: 9.

[005131 In an example, the amino acid sequence is SEQ ID NO: 10.

[005141 In an example, the amino acid sequence is SEQ ID NO: 11, [005151 In an example, the amino acid sequence is SEQ ID NO: 12.

[005161 In an example, the amino acid sequence is SEQ ID NO: 13.

[005171 In an example, the amino acid sequence is SEQ ID NO: 14.

[005181 In an example, the amino acid sequence is SEQ ID NO: 15.

[005191 In an example, the amino acid sequence is SEQ ID NO: 16.

[005201 In an example, the amino acid sequence is SEQ ID NO: 17.

[005211 In an example, the amino acid sequence is SEQ ID NO: 18.

[005221 In an example, the amino acid sequence is SEQ ID NO: 19.

[005231 In an example, the amino acid sequence is SEQ ID NO: 20.

[005241 In an example, the amino acid sequence is SEQ ID NO: 21.

1005251 In an example, the amino acid sequence is SEQ ID NO: 22.

[005261 In an example, the amino acid sequence is SEQ ID NO: 23.

[005271 In an example, the amino acid sequence is SEQ ID NO: 24.

[005281 In an example, the amino acid sequence is SEQ ID NO: 25.

[005291 In an example, the amino acid sequence is SEQ ID NO: 26.

[005301 In an example, the amino acid sequence is SEQ ID NO: 27.

[005311 In a Twelfth Aspect: The ligand of aspect 11, wherein the ligand is an antibody or antibody fragment. For example, the antibody or antibody fragment is a PCSK9 antagonist, eg, neutralises PCSK9, [005321 Examples of such antibodies are disclosed, for instance, in WO 2008/057457, WO2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/133647, WO 2009/100297, WO 2009/100318, WO 201 1/037791, WO 2W 1/053759, WO 201 1/053783, WO 2008/] 25623, WO 2011/072263, WO 2009/055783, WO 2010/0295 13, WO 2011/Il 1007, WO 2010/077854, the disclosures and sequences of such antibodies being incorporated herein for use in the invention in their entireties by reference. One specific example is AJVIG (Amgen), LY3015014 (Eli Lilly) or alirocumab, Advantageously, the ligand is or comprises alirocumab. Alternatively, the ligand is or comprises evolocumab.

[005331 In an example, the ligand is SAR236553/REGN727 (Sanofi Aventis/Regeneron) or a PCSK9-binding derivative thereof [005341 In an example, the ligand comprises or consists of a neutralizing antibody that binds to the PCSK9, wherein the antibody binds to PCSK9 and reduces the likelihood that PCSK9 binds to LDLR, [005351 The ligand of aspect t t, wherein the ligand is a PCSK9 antagonist. eg, neutralises PCSK9, [005361 In an example of any aspect of the invention, the ligand comprises or consists a ligand selected from evolocumab, IDOS-IgG2 (Merck & Co.), ALN-PCSO2 (Alnylam), RN316 (Pfizer-Rinat), LY3OISW4 (Eli Lilly) and alirocumab (SAR236553/REGN727; Sanofi Aventis/Regeneron).

[005371 In Thirteenth Aspect: The ligand of any one of aspects 1 to 10, wherein (i) the ligand comprises a sequence of contiguous nucleotides that specifically hybridises to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof or specifically hybridises to an antisense sequence or an RNA transcript of said sequence, wherein said sequence of contiguous nucleotides hybridises to at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28 or hybridises to an antisense sequence or an RNA transcript thereof respectively; and/or (ii) the ligand comprises a sequence of at least 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or is an antisense sequence or RNA version of said contiguous nucleotides, wherein said sequence of contiguous nucleotides comprises at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28.

[005381 In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37, These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

[005391 In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta c/ a! 2006).

[005401 In all example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen eta! 2005).

[005411 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

[005421 In an example, the nucleotide sequence is SEQ ID NO: 29.

[005431 In an example, the nucleotide sequence is SEQ ID NO: 30.

[005441 In an example, the nucleotide sequence is SEQ ID NO: 31.

[005451 In an example, the nucleotide sequence is SEQ ID NO: 32.

[005461 In an example, the nucleotide sequence is SEQ ID NO: 33.

[005471 In an example, the nucleotide sequence is SEQ ID NO: 34.

[005481 In an example, the nucleotide sequence is SEQ ID NO: 35.

[005491 In an example, the nucleotide sequence is SEQ ID NO: 36.

[005501 In an example, the nucleotide sequence is SEQ ID NO: 37.

[005511 In an embodiment, the ligand comprises at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or 100 contiguous nucleotides of said nucleotide sequence.

1005521 lii a Fourteenth Aspect: The ligand of any preceding aspect, wherein said disease or condition is hyperlipidaemia, hypercholesterolaemia (eg, familial hypercholesterolaemia), heart attack, stroke, coronary heart disease, atherosclerosis or a cardiovascular disease or condition.

1005531 The ligand of any preceding aspect, wherein the disease or condition is hypercholesterolemia, hyperlipidemia, hypercholesterolemia, dyslipidemia, cholestatic liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis or a cardiovascular disease.

1005541 In an example, said disease or condition is hypercholesterolaemia, The term "hypercholesterolaemia," as used herein, refers to a condition in which cholesterol levels are elevated above a desired level. In some embodiments, this denotes that serum cholesterol levels are elevated, In some embodiments, the desired level takes into account various "risk factors" that are known to one of skill in the art (and are described or referenced in US20120093818).

1005551 The ligand of any preceding aspect, wherein the human is identified as heterozygous for Familial Hypercholesterolemia, statin intolerant, statin uncontrolled, or at risk for developing hypercholesterolemia, dyslipidemia, cholestatic liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis or a cardiovascular disease.

1005561 In a Fifteenth Aspect: The ligand of any preceding aspect, wherein said disease or condition is associated with elevated LDL cholesterol.

1005571 Cholesterol levels are measured in milligrams (mg) of cholesterol per deciliter (dL) of blood in the United States and some other countries, Canada and most European countries measure cholesterol in millimoles (mmol) per liter (L) of blood, Below are general guideline ideal ranges and elevated ranges.

Total cholesterol Total cholesterol* (U.S. and some other countries) (Canada and most of Europe) Below 200 mg/dL Below 5,2 mmol/L Ideal 200-239 mg/dL 5.2-6.2 mmol/L Borderline high 240 mg/dL and above Above 6.2 mmol/L High LDL cholesterol LDL cholesterol* (U.S. and some other countries) (Canada and most of Europe) 100-129 mg/dL 2.6-3.3 mmol/L Ideal 30159 mg/dL 3.4-4.] mmol/L Borderline high 160-189 mg'dL 4.1-4.9 mmol/L High mg/dL and above Above 4,9 mmol/L Very high *Canadian and European guidelines differ slightly from U.S. guidelines. These conversions are based on U.S. guidelines.

[005581 Elevated LDL cholesterol is, therefore, 160 mg/dL or above (4,1 mmol/L or above).

[005591 In a Sixteenth Aspect: The ligand of any preceding aspect, wherein the ligand inhibits human PCSK9 binding to human LDL receptor and optionally has been or is determined as capable of such inhibition.

[005601 In an example, the method comprises (before administering the ligand) determining that the ligand is capable of such inhibition.

[005611 Inhibition determination is eg, inhibition in a blood or serum sample, at rtp, at ph7, at 37 degrees centigrade and/or under the physiological conditions of a human body.

[005621 In a Seventeeth Aspect: The ligand of any preceding aspect, wherein the human is resistant or substantially resistant to statin (eg, avorstatin and/or fluvastatin) treatment of said disease or condition, [005631 In an Eighteenth Aspect: The ligand of any preceding aspect, wherein the ligand is for treating and!or preventing a PC SK9-mediated disease or condition in a human (i) whose genome comprises SEQ ID NO: 29 and wherein the human is of ASW,YRI,GBR,TSI, CLM,LWK,MXL,JPT,PUR,IBS,FIN or CEU ancestry; or (ii) whose genome comprises SEQ ID NO: 30 and wherein the human is of ASW,YRI,GBR,TSI,CLM, CHB,LWK,CHS,JPT,PUR,FIN or CEU ancestry; or (iii) whose genome comprises SEQ ID NO: 32 and wherein the human is of ASW,GBR,TSI,CLM, JPT,PIJR,IBS,FIN or CEU ancestry; or (iv) whose genome comprises SEQ ID NO: 33 and wherein the human is of LWK,ASW,YRI or CLM ancestry; or (v)whose genome comprises SEQ ID NO: 34 and wherein the human is of LWK,ASW or YRI ancestry; or (vi) whose genome comprises SEQ ID NO: 35 and wherein the human is of PUR,TSI,F1IN or CEU ancestry; or (vii) whose genome comprises SEQ ID NO: 36 and wherein the human is of LWK,ASW or YRI ancestry; or (viii) whose genome comprises SEQ ID NO: 37 and wherein the human is of CHS,ASW,JPT,PUR or CFIB ancestry.

[005641 In a Ninteenth Aspect: The ligand of any preceding aspect, wherein the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human (i) that expresses PCSK9 form f and wherein the human is of ASW,YRI,GBR,TSI,CLM,LWK,MXL,JPT,PUR,IBS,FIN or CPU ancestry; or (ii) that expresses PCSK9 form c and wherein the human is of ASW,YRI,GBR,TSI,CLM,CHB,LWK,CUS,JPT,PUR,FIN or CPU ancestry; or (iii) that expresses PCSK9 fonnp and wherein the human is of ASW,GBR,TSI,CLM,JPT,PURJBS,FIN or CEU ancestry; or (iv) that expresses PCSK9 form rn and wherein the human is of LWK,ASW,YRT or CLM ancestry; or (v) that expresses PCSK9 form e and wherein the human is of LWK,ASW or YRI ancestry; or (vi) that expresses PCSK9 form hand wherein the human is of PIJR,TSI,FIN or CEU ancestry; or (vii) that expresses PCSK9 form aj and wherein the human is of LWK,ASW or YRI ancestry; or (viii) that expresses PCSK9 form q and wherein the human is of CHS,ASW,JPT,PUR or CUB ancestry.

[005651 In an example, said forms are the mature forms.

[005661 lii an example, said forms are the pro-forms.

[005671 In a Twentieth Aspect: A pharmaceutical composition or kit for treating and/or preventing a PCSK9-mediated condition or disease (eg, as recited in aspect 14 or 15), the composition or kit comprising a ligand of any preceding aspect and optionally a statin (eg, cerovastatin, atorvastatin, simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin or pravastatin); and optionally in combination with a label or instructions for use to treat and/or prevent said disease or condition in a human (eg, covering treatment of a human as recited in aspect 18 or 19); optionally wherein the label or instructions comprise a marketing authorisation number (eg, an FDA or EMA authorisation number); optionally wherein the label or instmctions comprise directions to administer alirocumab or evolocumab to said human; optionally wherein the kit comprises an TV or injection device that comprises the ligand (and, eg, also a statin).

[005681 In a Twenty-first Aspect: A method of producing an anti-human PCSK9 antibody binding site, the method comprising obtaining a plurality of anti-PC SK9 antibody binding sites, screening the antibody binding sites for binding to a human PC 51(9 selected from the group consisting of forms/ c, i p, in, e, h, ctj and q or a catalytic or C-terminal domain or a peptide thereof that comprises amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3 and isolating an antibody binding site that binds in the screening step, and optionally producing a form f c, r, p, in, e, h, aj or q PCSK9-binding fragment or derivative of the isolated antibody.

1005691 In an example, said forms are the mature forms.

1005701 In an example, said forms are the pro-forms.

1005711 lii an example of this and the next aspect, the plurality of binding sites comprises or consists of a plurality of 4-chain antibodies or fragments thereof eg, dAbs, Fabs or scFvs, Suitable methods for producing pluralities of binding sites for screening include phage display (producing a phage display library of antibody binding sites), ribosome display (producing a ribosome display library of antibody binding sites), yeast display (producing a yeast display library of antibody binding sites), or immunisation of a non-human vertebrate (eg, a rodent, eg, a mouse or rat, eg, a VelocimousetmT, Kvmousetmt, Xenomousemt, Aliva Mouse'TM, HuMab Mouse1, or MeMo 05M) with a PCSK9 epitope and isolation of a repertoire of antibody-producing cells (eg, a B-cell, plasma cell or plasmablast repertoire) and/or a repertoire of isolated antibodies.

1005721 In an example, the method comprises selecting one or more antibody binding sites that each specifically binds to a human PCSK9 epitope comprising amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3.

1005731 In a Twenty-second Aspect: A method of producing an anti-human PCSK9 antibody, the method comprising immunising a non-human vertebrate (eg, a mouse or a rat) with a human PCSK9 comprising an amino acid sequence selected from the group consisting of the amino acid sequences of forms f c, r, p, in, e, Ii, qj and q or a catalytic or C-terminal domain or a peptide thereof that comprises amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3 and isolating an antibody that binds a human PCSK9 comprising selected from the group consisting of forms.f c, i p, in, e, h, cii and q or a catalytic or C-terminal domain or a peptide thereof that comprises amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3, and optionally producing a form f c, r, p, in, e, h, cif or q PCSK9-binding fragment or derivative of the isolated antibody.

1005741 In an example, said forms are the mature forms.

1005751 In an example, said forms are the pro-forms.

[005761 In a Twenty-third Aspect: The method of aspect 21 or 22, comprising the step of obtaining a nucleic acid encoding the antibody, fragment, derivative or binding site and optionally inserting the nucleic acid in an expression vector.

[005771 For example, the method comprises isolating a cell (eg, B-cell, plasmablast, plasma cell or memory cell) comprising the nucleic acid, wherein the cell is obtained from a non-human vertebrate that has been immunised with the PCSK9 epitope.

[005781 In a Twenty-fourth Aspect: A kit for PCSK9 genotyping a human, wherein the kit comprises a nucleic acid (i) comprising a sequence of 10 or more (eg, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) contiguous nucleotides that specifically hybridises to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain-or C-terminal domain-encoding sequence thereof or specifically hybridises to an antisense sequence or an RNA transcript of said sequence, wherein said sequence of contiguous nucleotides hybridises to at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28 or hybridises to an antisense sequence or an RNA transcript thereof and!or (ii) comprising a sequence of at least 10 or more (eg, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-3 7 or comprising an antisense sequence or RNA version of said contiguous nucleotides, wherein said sequence of contiguous nucleotides comprises at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28.

[005791 In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37, These are naturally-occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above. These groups comprise variants that are associated with elevated LDL-C.

[005801 In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta c/ at 2006).

[005811 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen etaI200S).

[005821 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

[005831 In an example, the nucleotide sequence is SEQ ID NO: 29.

[005841 In an example, the nucleotide sequence is SEQ ID NO: 30.

[005851 In an example, the nucleotide sequence is SEQ ID NO: 31.

[005861 In an example, the nucleotide sequence is SEQ ID NO: 32, [005871 In an example, the nucleotide sequence is SEQ ID NO: 33.

[005881 In an example, the nucleotide sequence is SEQ ID NO: 34.

[005891 In an example, the nucleotide sequence is SEQ ID NO: 35.

[005901 In an example, the nucleotide sequence is SEQ ID NO: 36.

[005911 In an example, the nucleotide sequence is SEQ ID NO: 37.

[005921 In a Twenty-fifth Aspect: A kit for PCSK9 genotyping or phenotyping a human, wherein the kit comprises a ligand according to any one of aspects I to 19 or an antibody, fragment or derivative produced by the method of any one of aspects 21 to 23, [005931 In a Twenty-sixth Aspect: Use of an anti-PC 51(9 ligand that binds a human PCSK9 selected from the group consisting of forms/ c, i p, iii, e, h, aj and q in the manufacture of a m edicament for treating and/or preventing a PC 5K9-m edi ated disease or condition in a human whose genome comprises a nucleotide sequence selected from the oup consisting of SEQ ID NOs: 29-3 7, optionally for treating and/or preventing a PCSK9-mediated disease or condition in a human as recited in aspect 18 or 19, [005941 In an example, said forms are the mature forms.

[005951 In an example, said forms are the pro-forms.

[005961 In a Twenty-seventh Aspect: Use of an anti-PC 5K9 ligand that binds a human PCSK9 selected from the group consisting offormsf c, r, p iii, e, h, aj and qin the manufacture of a medicament for targeting said PC SK9 in a human to treat and/or prevent a disease or condition mediated by PCSK9, optionally for targeting PCSK9 in a human as recited in aspect 18 or 19.

[005971 In an example, said forms are the mature forms.

[005981 In an example, said forms are the pro-forms.

[005991 In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37.

[006001 In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciofta e a! 2006).

[006011 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen eta! 2005).

[006021 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32,34,35,36 and 37; or selected from the group consisting of SEQ ID NOs: 3], 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

[006031 In an example, the nucleotide sequence is SEQ ID NO: 29.

[006041 In an example, the nucleotide sequence is SEQ ID NO: 30.

[006051 In an example, the nucleotide sequence is SEQ ID NO: 31.

[006061 In an example, the nucleotide sequence is SEQ ID NO: 32, [006071 In an example, the nucleotide sequence is SEQ ID NO: 33.

[006081 In an example, the nucleotide sequence is SEQ ID NO: 34.

[006091 In an example, the nucleotide sequence is SEQ ID NO: 35.

[006101 In an example, the nucleotide sequence is SEQ ID NO: 36.

[006th In an example, the nudeotide sequence is SEQ ID NO: 37.

[006121 The ligand carl be any anti-PCSK9 ligand disclosed herein.

[006131 In a Twenty-eight Aspect: The use of aspect 26 or 27, wherein the ligand, human, disease or condition is according to any one of aspects to]9.

[006t41 In a Twenty-ninth Aspect: A method of targeting a PCSK9 for treating and/or preventing a PCSK9-mediated disease or condition in a human, the method comprising administering an anti-PCSK9 ligand to a human comprising a nucleotide sequence selected from the group consisting SEQ ID NOs: 29-3 7, whereby a PCSK9 encoded by said nucleotide sequence is targeted.

[006151 The ligand can be any anti-PCSK9 ligand disclosed herein.

[006161 In a Thirtieth Aspect: The method of aspect 29, wherein the method comprises targeting a human PCSK9 selected from the group consisting of forms f c, ,, p, in, e, h, aj and q with said ligand to treat and/or prevent said disease or condition in said human.

[006171 In an example, said forms are the mature forms.

[006 181 In an example, said forms are the pro-forms.

[006191 In a Thirty-first Aspect: A method of treating and/or preventing a disease or condition mediated by PCSK9 in a human, the method comprising targeting a human PCSK9 selected from the group consisting offormsf C, r, p, iii, e, h, aj and q by administering to the human a ligand that binds said PCSK9 thereby treating and/or preventing said disease or condition in the human.

[006201 In an example, said forms are the mature forms.

[006211 In an example, said forms are the pro-forms.

[006221 The ligand can be any anti-PCSK9 ligand disclosed herein.

[006231 In a Thirty-second Aspect: The method of aspect 31, wherein the genome of the human comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37.

[006241 In an example, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or selected from the group consisting of SEQ ID NOs: 29-32 and 34-37; or selected from the group consisting of SEQ ID NOs: 29-32, 34, 35 and 37.

[006251 lii an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta eta! 2006).

[006261 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, that encode 670G which is a marker for severity of coronary atherosclerosis (Chen ci at 2005), [006271 In an example, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allele (haplotype) sequences that have a naturally-occurring combination of differences from SEQ ID NO: 28 (form a) and which meet the criteria set out above.

[006281 In an example, the nucleotide sequence is SEQ ID NO: 29.

[006291 In an example, the nucleotide sequence is SEQ ID NO: 30, [006301 In an example, the nucleotide sequence is SEQ ID NO: 3].

[006311 In an example, the nucleotide sequence is SEQ ID NO: 32.

[006321 In an example, the nucleotide sequence is SEQ ID NO: 33.

[006331 In an example, the nucleotide sequence is SEQ ID NO: 34.

1006341 In an example, the nucleotide sequence is SEQ ID NO: 35, 1006351 In an example, the nucleotide sequence is SEQ ID NO: 36.

[006361 In an example, the nucleotide sequence is SEQ ID NO: 37.

[006371 In a Thirty-third Aspect: The method of any one of aspects 29 to 32, wherein the human has been or is genotyped as positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic-or C-terminal domain-encoding sequence thereof.

[006381 In a Thirty-fourth Aspect: The method of any one of aspects 29 to 33, wherein the human has been or is phenotyped as positive for a human PCSK9 selected from the group consisting of forms f c, r, p, in, e, h, a/and q.

[006391 In an example, said forms are the mature forms.

[006401 In an example, said forms are the pro-forms.

[006411 Iii a Thirty-fifth Aspect: The method of any one of aspects 29 to 34, wherein the method comprises genotyping the human as positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-3 7 or the catalytic-or C-terminal domain-encoding sequence thereof [006421 In a Thirty-sixth Aspect: The method of any one of aspects 29 to 35, wherein the method comprises phenotyping the human as positive for a human PCSK9 sequence selected from the group consisting of forms / c, r, p, rn, e, h, qj and q.

[006431 In an example, said forms are the mature forms.

[006441 In an example, said forms are the pro-forms.

[006451 In a Thirty-seventh Aspect: The method of any one of aspects 29 to 36, wherein the human has been or is genotyped as heterozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic-or C-terminal domain-encoding sequence thereof optionally wherein the human has been or is genotyped as comprising the nucleotide sequence of SEQ ID NO: 28 or the catalytic-or C-terminal domain-encoding sequence thereof and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-3 7 or the catalytic-or C-terminal domain-encoding sequence thereof [006461 In a Thirty-eighth Aspect: The method of any one of aspects 29 to 37, wherein the genome of the human has been or is genotyped as homozygous for a nucleotide sequence selected from the group consisting of of SEQ ID NOs: 29-37 or the catalytic-or C-terminal domain-encoding sequence thereof [006471 In a Thirty-ninth Aspect: The method of any one of aspects 29 to 38, wherein the method comprises genotyping the human for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic-or C-terminal domain-encoding sequence thereof before administering the ligand to the human, wherein the ligand is determined to be capable of binding to a PCSK9 encoded by said selected sequence.

1006481 In a Fortieth Aspect: The method of any one of aspects 29 to 39, wherein the ligand, human, disease or condition is according to any one of aspects 1 to t 9.

1006491 In a Forty-first Aspect: A method according to any one of aspects 29 to 40 for treating and/or preventing a condition or disease as recited in aspect 14 or 15, the method comprising administering said ligand and a statin (eg, cerovastatin, atorvastatin, simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin or pravastatin) to the human.

[006501 In a Forty-second Aspect: The method of aspect 41, wherein the ligand and statin are administered separately.

1006511 In a Forty-third Aspect: The method of aspect 41, wherein the ligand and statin are administered simultaneously.

[006521 In a Forty-fourth Aspect: The method of any one of aspects 29 to 43, wherein the ligand is administered by subcutaneous injection.

1006531 In a Forty-fifth Aspect: A method of PCSK9 genotyping a nucleic acid sample of a human, the method comprising identifying in the sample the presence of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic-or C-terminal domain-encoding sequence thereof 1006541 In a Forty-sixth Aspect: A method of PCSK9 typing a protein sample of a human, the method comprising identifying in the sample the presence of a human PCSK9 selected from the group consisting of forms f c, i p, in, e, h, aj and q.

1006551 In an example, said forms are the mature forms.

1006561 In an example, said forms are the pro-forms.

1006571 In a Forty-seventh Aspect: The method of aspect 45 or 46, comprising obtaining a sample of serum, blood, faeces, hair, tissue, cells, urine or saliva from a human, whereby the nucleic acid or protein sample is obtained and used in the step of identifying said sequence.

[006581 In a Forty-eighth Aspect: The method of any one of aspects 45 to 47, comprising using a ligand according to any one of aspects t to 19 to carry out said identifying step.

1006591 In a Forty-ninth Aspect: A method of treating and/or preventing in a human patient a cardiovascular disease or condition, or a disease or condition that is associated with elevated LDL cholesterol (eg, hypercholesterolaemia), wherein the patient is receiving or has previously received statin treatment for said disease or condition, the method comprising typing the patient using a method of any one of aspects 45 to 48 and administering a ligand according to one of aspects 1 to 19 whereby the human is treated or said disease or condition is prevented; optionally also reducing or stopping statin treatment.

1006601 In an example, said reducing or stopping comprises reducing the dose and/or dosing frequency of statin.

1006611 In a Fiftieth Aspect: A diagnostic, therapeutic or prophylactic kit comprising a ligand that is capable of binding to or has been or is determined as capable of binding to an amino acid sequence selected from SEQ ID NOs: 4-27 and instructions for carrying out the method of any one of aspects 46 to 49 and/or a label or instructions indicating or covering administration of the ligand to a human as defined in any one of aspects ito 19.

1006621 In a Fifty-first Aspect: A diagnostic, therapeutic or prophylactic kit comprising a nucleic acid probe comprising anucleotide sequence that specifically hybridises to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or an anti sense sequence or RNA transcript thereof and instructions for carrying out the method of aspect 45, 47 or 48.

1006631 In examples of the present invention, the ligand specifically binds to human PCSK9, eg, one or more of the rare PCSK9 variants disclosed herein (eg, one, two, three, more or all mature forms[ c, r, p, in, e, h, aj and q) and optionally also the a and!or a' form.

For example, the ligand specifically binds to mature form [and/or c as well as form a.

Determination of such binding can be performed by any antibody binding test as known in the art, eg, by surface plasmon resonance. Binding to each such form is, for example, respectively with a Kd of at least I mlvi, t OOnM, 1 nM, 100pM, 10pM or 1pM.

1006641 In an example, the ligand binds form a and a PCSK9 selected from the group consisting of forms[ c, i p, in, e, h, cii and q, wherein the ligand binding to said selected form is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms, In an embodiment, both forms are pro-forms.

1006651 In an example, the ligand binds form a and formf wherein the ligand binding to formf is with a lCd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms, In an embodiment, both forms are pro-forms.

1006661 In an example, the ligand binds form a and form c, wherein the ligand binding to form c is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the lCd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms.

[006671 In an example, the ligand binds form a and form r, wherein the ligand binding to form r is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms.

[006681 In an example, the ligand binds form a and form p, wherein the ligand binding to form p is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms, In an embodiment, both forms are pro-forms.

[006691 In an example, the ligand binds form a and form m, wherein the ligand binding to form in is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms, In an embodiment, both forms are pro-forms.

[006701 In an example, the ligand binds form a and form e, wherein the ligand binding to form e is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms, In an embodiment, both forms are pro-forms, [006711 In an example, the ligand binds form a and form h, wherein the ligand binding to form his with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95?/'b of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms, [006721 In an example, the ligand binds form a and form aJ, wherein the ligand binding to form aj is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms, [006731 In an example, the ligand binds form a and form q, wherein the ligand binding to form q is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the lCd for binding to form a. In an embodiment, both forms are mature forms. In an embodiment, both forms are pro-forms, [006741 In examples of the present invention, the ligand neutralises human PCSK9, eg, one or more of the rare PCSK9 variants disclosed herein (eg, one, two, three, more or all mature formsf c, r, p, in, e, h, aj and q) and optionally also the a and/or a' form. For example, the ligand neutralises mature form fand'ór C as well as/brin a. Determination of neutralisation can be performed, for example, by any neutralisation assay method disclosed in US2O 12009381 8A1 (Amgen, mc) or US2O 11 0065902A1 (Regeneron Pharmaceuticals, mc). Ligands of the invention that bind or target PCSK9 are useful, for example, for therapeutic and prophylactic applications disclosed in US20120093818A1 and US2O1 10065902A1, these specific disclosures being incorporated herein by reference for use in the present invention and for possible inclusion in claims herein.

[006751 In embodiments where the ligand is used for therapeutic applications, an antigen binding protein can inhibit, interfere with or modulate one or more biological activities of a PCSK9 (eg, one or more of the rare variants disclosed herein and optionally also the a and/or a form). In one embodiment, ligand binds specifically to human PCSK9 (eg, one or more of the rare variants disclosed herein and optionally also the a and/or a' form) and/or substantially inhibits binding of human PCSK9 (eg, said one or more of the rare variants disclosed herein and optionally also the a and/or a form) to LDLR by at least 20%, eg, 20%- 40%, 40-60%, 60-80%, 80-85%, or more (for example, by measuring binding in an in vitro competitive binding assay). In an example, the ligand is an antibody.

[006761 In an embodiment, the ligand has a Kd of less (binding more tightly) than I o-, 10-8, 1 o9, 10_ID, 10H, ]o_12 I o' M for binding to one, two or more of the rare variants disclosed herein and optionally also the a and/or a form. In an example, Kd is determined using SPR, [006771 In an embodiment, the ligand has an ICSO for blocking the binding of LDLR to one or more of the rare PCSK9 variants disclosed herein (and optionally also the a and/or a' form) of less than 1 microM, 1000 nM to tOO nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, lOOpMto 10pM, lOpMto 1pM, [006781 In an embodiment, the ligand has an ICSO for blocking the binding of LDLR to the a and/or a'form ofPCSK9 that is no more than 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10-fold more (ie, more inhibitory) than the 1C50 for blocking the binding of LDLR to one or more of the rare PCSK9 variants disclosed herein (eg, one or more PC 51(9 proteins comprising a sequence selected from SEQ ID NOs: 4 to 27). Additionally or alternatively, for example, the ligand has an 1C50 for blocking the binding of LDLR to (i) the a and/or a' form ofless than I microM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to I nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, pM to 10 pM, 10 pMto 1 pM, eg, in the range of 1mM to 1pM (eg, 1mM to 100pM; lOnM to 100pM; tnM to 10pM; or tOOpM to 1pM) and (ii) one or more PCSK9 proteins comprising a sequence selected from SEQ ID NOs: 4 to 27 of less than I microM, 1000 nM tolOOnM, lOOnMtolOnM, l0ntvitolnM,l000pMtoSOOpM,SOOpMto200pM,less than 200 pM, 200 pMto 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, eg, in the range of 1mM to 1pM (eg, 1mM to 100pM; iOnM to 100pM; mM to 10pM; or 100pM to 1pM).

1006791 In an embodiment, the ligand binds to the a and/or a' fonn of PCSK9 with a binding affinity (1(d) that is greater than up to 10%, greater than up to 20%, greater than up to 40%, greater than up to 50%, greater than up to 55%, greater than up to 60%, greater than up to 65%, greater than up to 70%, greater than up to 75%, greater than up to 80%, greater than up to 85%, greater than up to 90%, greater than up to 95% or greater than up to 100% (ie, is double) relative to binding to a PCSK9 comprising a sequence selected from SEQ ID NOs: 4 to 27. Such binding measurements can be made using a variety of binding assays known in the art, eg, using surface plasmon resonance (SPR), such as by Biacore'TM or using the ProteOn XPR36Thf (Bio-Rad®), or using KinExA® (Sapidyne Instmments, mc).

[006801 In one embodiment, the surface plasmon resonance (SPR) is carried out at 25°C.

in another embodiment, the SPR is carried out at 37°C.

[006811 In one embodiment, the SPR is carried out at physiological pH, such as about pt-17 or at pH7,6 (eg, using Hepes buffered saline at pH7,6 (also referred to as HBS-EP)).

[006821 lii one embodiment, the SPR is carried out at a physiological salt level, eg, 150mM NaCI, [006831 In one embodiment, the SPR is carried out at a detergent level of no greater than 0.05% by volume, eg, in the presence of P20 (polysorbate 20; eg, Tween-2OTM) at 0.05% and EDTA at 3mM, [006841 In one example, the SPR is carried out at 25°C or 37°C in abufferat pH7.6, 1 50mM NaC1, 0.05% detergent (eg, P20) and 3mM EDTA. The buffer can contain 10mM Hepes. In one example, the SPR is carried out at 25°C or 37°C in HBS-EP. HBS-EP is available from Teknova Inc (California; catalogue number H8022).

[006851 In an example, the affinity of the ligand which is an antibody is determined using SPRby 1. Coupling anti-mouse (or other relevant vertebrate) IgG (eg, Biacore BR-1008-38) to a biosensor chip (eg, GLM chip) such as by primary amine coupling; 2. Exposing the anti-mouse IgG (vertebrate antibody) to a test IgG antibody to capture test antibody on the chip; 3. Passing the test antigen over the chip's capture surface at 024nM, 2SônM, 64nM, l6nM, 4nM with a OnM (i.e. buffer alone); and 4. And determining the affinity of binding of test antibody to test antigen using surface plasmon resonance, eg, under an SPR condition discussed above (eg, at 25°C in physiological buffer). SPR can be carried out using any standard SPR apparatus, such as by BiacoreTM or using the ProteOn XPR36TM (Bio-Rad®).

[006861 Regeneration of the capture surface can be carried out with 10mM glycine at pHI.7. This removes the captured antibody and allows the surface to be used for another interaction, The binding data can be fitted to 1:1 model inherent using standard techniques, eg, using a model inherent to the ProteOn XPR36" analysis software.

[006871 h an embodiment, assaying or testing of a ligand of the invention is carried out at or substantially at pH7 (eg, for in vitro tests and assays) and at or substantially at rtp.

[006881 One example of an IgG2 heavy chain constant domain of an anti-PCSK9 antibody of the present invention has the amino acid sequence as shown in SEQ ID NO: 154, FIG. 3KK of US201200938t8A1, which sequence is incorporated herein by reference.

[006891 One example of an lgG4 heavy chain constant domain of an anti-PC 5K9 antibody of the present invention has the amino acid sequence as shown in SEQ ID NO: 155, FIG. 3KK ofUS201200938t8A1, which sequence is incorporated herein by reference.

[006901 One example of a kappa light chain constant domain of an anti-PCSK9 antibody has the amino acid sequence as shown in SEQ ID NO: 57, FIG, 3KK which sequence is incorporated herein by reference.

[006911 One example of a lambda light chain constant domain of an anti-PCSK9 antibody has the amino acid sequence as shown in SEQ ID NO: 156, FIG, 31(1K of US2O 1200938] 8A], which sequence is incorporated herein by reference.

[006921 In examples of the present invention, the ligand binds mature PCSK9, eg, a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or t'form.

[006931 In examples of the present invention, the ligand binds the catalytic domain of PC SIC, eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or form, [006941 In examples of the present invention, the ligand binds the prodomain of PCSK9, eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a' form.

1006951 In some embodiments, the ligand binds to the V domain of PCSK9, eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a' form. In some embodiments, the ligand binds to the V domain of PCSK9 (eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a' form) and prevents (or reduces, eg, by at least 10%) PC SK9 from binding to LDLR, In some embodiments, the ligand binds to the V domain of PCSK9 (eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a'form), and while it does not prevent (or reduce) the binding of PCSK9 to LDLR, the ligand prevents or reduces (eg, by at least 10%) the adverse activities mediated through PCSK9 on LDLR.

1006961 In examples of the present invention, the ligand is or comprises a fully human antibody. In an example, the ligand comprises human variable regions or humanised variable regions.

1006971 In an example, the ligand of the invention specifically binds to an epitope of a human PCSK9 selected from the group consisting offormsf c, r, p, m, e, h, q/ and q, wherein the epitope comprises at least one amino acid that is not found in form a. For example, the amino acid is selected from the group consisting of 46L, 53V, 4255, 4431, 474V, 619P and 670G (numbering as used in SEQ ID NO:]). For example, the amino acid is selected from the group consisting of 425S, 4431, 474V, 619P and 670G (numbering as used in SEQ ID NO: I). For example, the amino acid is selected from the group consisting of 425S and 443T (numbering as used in SEQ ID NO:]). For example, the amino acid is selected from the group consisting of 474V, 619P and 670G (numbering as used in SEQ ID NO: 1). In an example, the PCSK9 form is the mature form. In an example, the PCSK9 form is the pro-form, In an example, the ligand also specifically binds to form a and/or a'. In an embodiment, the ligand specifically binds to an epitope offormfPCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form c PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to an epitope of form r PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope offormp PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form m PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form af PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form qPCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.

1006981 In an embodiment, ligand binds specifically to the pro-domain of a human PCSK9 selected from the group consisting offormsf c, r, p, m, e, h, aj and q. In an example, the ligand also specifically binds to the pro-domain of form a and/or a', In an embodiment, the ligand specifically binds to the pro-domain of formfPCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form c PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the pro-domain of form r PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the pro-domain of form P PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form in PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the pro-domain of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the pro-domain of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form aj PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the pro-domain of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.

[006991 In an embodiment, ligand binds specifically to the catalytic domain of a human PCSK9 selected from the group consisting offormsf c, i p, in, e, h, aj and q. In an example, the ligand also specifically binds to the catalytic domain of form a and/or a'. In an embodiment, the ligand specifically binds to the catalytic domain of form [PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the catalytic domain of form c PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form r PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form p PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form in PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, 1i an embodiment, the ligand specifically binds to the catalytic domain of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the catalytic domain of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form qj PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.

1007001 In an embodiment, ligand binds specifically to the C-terminal domain of a human PCSK9 selected from the group consisting offormsf c, i p, iii, e, h, aj and q. In an example, the ligand also specifically binds to the C-terminal domain of form a and/orci', In an embodiment, the ligand specifically binds to the C-terminal domain of form fPCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, in an embodiment, the ligand specifically binds to the C-terminal domain of form c PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the C-terminal domain of form r PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the C-terminal domain offormp PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the C-terminal domain of form m PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the C-terminal domain of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the C-terminal domain of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form a/ PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the C-terminal domain of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.

[007011 In an embodiment, ligand binds specifically to the substrate-binding groove of a human PCSK9 selected from the group consisting of formsf c, i p, rn, e, h, aj and q (see Cunningham et al., Nat Stmct Mol Biol. 2007 May; 14(5):413-9. Epub 2007 Apr 15, "Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia", incorporated herein in its entirety by reference). In an example, the ligand also specifically binds to the substrate-binding groove of form a and/or a', In an embodiment, the ligand specifically binds to the Substrate-binding groove of form fPCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the Substrate-binding groove of form c PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form r PC SK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the Substrate-binding groove of form p PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the Substrate-binding groove of form in PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, in an embodiment, the ligand specifically binds to the Substrate-binding groove of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the Substrate-binding groove of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the Substrate-binding groove of form aj PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, In an embodiment, the ligand specifically binds to the Substrate-binding groove of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a, [007021 Reference is made to US20120093818A1 (Amgen, mc), the entire disclosure of which is incorporated herein, This patent application discloses relevant ligands for use in the present invention, as well as examples and methods of producing and testing ligands that can be used with reference to the present invention.

[007031 In an example, the ligand is or comprises an antibody disclosed in Table 2 of US20120093818A1 (Amgen, mc) or is a PCSK9-binding derivative thereof [007041 In an embodiment, the PCSK9-binding ligand of the invention is selected from the antigen binding proteins disclosed in US20120093818A1 (Amgen, mc), eg, in paragraphs [00091 to [00141 and [0058] to [0063] ofUS20120093818Al; all of these disclosures (including the sequences of such proteins) are incorporated herein by reference as though explicitly recited herein and for possible inclusion in one or more claims or for use in the present invention.

[007051 In this paragraph SEQ ID NOs are those as appearing in U520120093 8 t 8A1 (Amgen, mc) and these sequences are incorporated herein by reference as though explicitly recited herein and for possible inclusion in one or more claims or for use in the present invention. In some aspects, the ligand of the invention comprises an isolated antigen binding protein that binds PCSK9 comprising: A) one or more heavy chain complementary determining regions (CDRI-ls) selected from the group consisting of: (i) a CDRI-1] from a CDREII in a sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; (ii) a CDRH2 from a CDRTI2 in a sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 5, 53, 48, 54, 55, 56, 49, 57, 50, 9], 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; (iii) a CDRH3 from a CDRH3 in a sequence selected from the group consisting of SEQ ID NO: 74, 85, 7 t, 72, 67, 87, 58, 52, 5 t, 53, 48, 54, 55, 56, 4, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 6c,81, and 60; and (iv) a CDRH of(i), (ii), and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 4 amino acids; B) one or more light chain complementary determining regions (CDRL5) selected from the group consisting of (i) a CDRL1 from a CDRL1 in a sequence selected from the group consisting of SEQ ID NO: 5, 7,9, 10, 12, 13, ]5, ]6, F/, 18, 19, 20, 2], 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (ii) a CDRL2 from a CDRL2 in a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, t2, t3, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (iii) a CDRL3 from a CDRL3 in a sequence selected from the group consisting of SEQ ID NO: 5,7,9, 10, ]2, 3, ]5, 6, 17, 18, 19, 20, 2], 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; and (iv)a CDRL of(i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 4 amino acids; or C) one or more heavy chain CDRI-ls of A) and one or more light chain CDRLs of B). In some embodiments, the isolated antigen binding protein comprises at least one CDRH of A) and at least one CDRL of B). In some embodiments, the isolated antigen binding protein comprises at least two CDRH of A) and at least two CDRL of B). In some embodiments, the isolated antigen binding protein comprises said CDRH, CDRH2, CDRH3, CDRL 1, CDRL2 and CDRL3. In some embodiments, the CDRH of A) is selected from at least one of the group consisting of: (i) a CDRH1 amino acid sequence selected from the CDRH1 in a sequence selected from the group consisting of SEQ ID NO: 67, 79, 89, and 49; (ii) a CDRH2 amino acid sequence selected from the CDRH2 in a sequence selected from the group consisting of SEQ ID NO: 67, 79, 89, and 49; (iii) a CDRH3 amino acid sequence selected from the CDRH3 in a sequence selected from the group consisting of SEQ ID NO: 67, 79, 89, and 49; and (iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids. In addition, the CDRL of B) is selected from at least one of the group consisting of: (i) a CDRL amino acid sequence selected from the CDRL1 in a sequence selected from the group consisting of SEQ ID NO: 12, 35, 32, and 23; (ii) a CDRL2 amino acid sequence selected from the CDRL2 in a sequence selected from the group consisting of SEQ ID NO: 12, 35, 32, and 23; (iii) a CDRL3 amino acid sequence selected from the CDRL3 in a sequence selected from the group consisting of SEQ ID NO: t2, 35, 32, and 23; and (iv)a CDRL of(i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids; or C) one or more heavy chain CDRI-ls of A) and one or more light chain CDRLs of B, In some embodiments, the CDRH of A) is selected from at least one of the group consisting of: (i) a CDRH1 amino acid sequence of the CDRH1 amino acid sequence in SEQ ID NO: 67; (H) a CDRH2 amino acid sequence of the CDRH2 amino acid sequence in SEQ ID NO: 67; (iii) a CDRH3 amino acid sequence of the CDRH3 amino acid sequence in SEQ ID NO: 67; and (iv) a CDR}I of(i), (H) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids; said CDRL of B) is selected from at least one of the group consisting of: (i) a CDRLI amino acid sequence of the CDRL1 amino acid sequence in SEQ ID NO: 12; (H) a CDRL2 amino acid sequence of the CDRL2 amino acid sequence in SEQ ID NO: 12; (Hi) a CDRL3 amino acid sequence of the CDRL3 amino acid sequence in SEQ ID NO: 12; and (iv) a CDRIL of(i), (H) and (Hi) that contains one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids; or C) one or more heavy chain CDRHs of A) and one or more light chain CDRLs of B). In some embodiments, the antigen binding protein comprises A) a CDRHt of the CDRHI sequence in SEQ ID NO: 67, a CDRH2 of the CDRI-12 sequence in SEQ ID NO: 67, and a CDRH3 of the CDRH3 sequence in SEQ ID NO: 67, and B) a CDRLt of the CDRL1 sequence in SEQ ID NO: 12, a CDRL2 of the CDRL2 sequence in SEQ ID NO: 12, and a CDRL3 of the CDRL3 sequence in SEQ ID NO: t2. In some embodiments, the antigen binding protein comprises a heavy chain variable region (VU) having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 8t, and 60, and/or alight chain variable region (VL) having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some embodiments, the VII has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 4, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 6c,81, and 60, and/or the VL has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 2], 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46, In some embodiments, the VH is selected from the group consisting of SEQ ID NO: 74, 85, 7], 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or the VL is selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, ]2, ]3, 5, 16, 17, ]8, ]9, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46.

[007061 In an example of any aspect of the invention, the PCSK9-targeting or binding ligand comprises or consists ofATVlGl4S or 3 1H4, 16F12, I IF 1, 8A3 or 21B 12 disclosed in US20120093818A1 (Amgen, mc) or an antibody comprising the variable domains of AMG] 45, 3 F14, 6F 12, II F], 8A3 or 21 B 12, the disclosures of which (including sequences) are incorporated herein by reference as though explicitly recited herein and for possible inclusion in one or more claims or for use in the present invention, Preferably, the PCSK9-targeting or binding ligand comprises or consists of AMG] 45.

[007071 In an example, the A1v10145 or other ligand of the invention is glycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos or Hek293 cell). In an example, the ligand of the invention is produced in CHO.

[007081 Reference is made to 1J5201 I 0065902A] (Regeneron Pharmaceuticals, mc), the entire disclosure of which is incorporated herein. This patent application discloses relevant ligands for use in the present invention, as well as examples and methods of producing and testing ligands and determining medical efficacy that can be used with reference to the present invention.

[007091 Reference is made to the following PCT applications, the entire disclosures of which are incorporated herein, These disclose relevant ligands for use in the present invention, as well as examples and methods of producing and testing ligands and determining medical efficacy that can be used with reference to the present invention.

[007101 Antibody ligands to PCSK9 are described in, for example, WO 2008/057457, WO 2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/125623, and US 2008/0008697; each of which is incorporated by reference herein in its entirety..

[007111 lii an example, the ligand is or comprises an antibody disclosed in the Examples ofUS2Ot t0065902Ai (eg, 316P or 30DM) or is a PCSK9-binding derivative thereof All of these disclosures (including the sequences of such proteins and corresponding nucleotide sequences) are incorporated herein by reference as though explicitly recited herein and for possible inclusion in one or more claims or for use in the present invention, In an embodiment, the ligand is or comprises the variable domains of antibody 3 16P or 300N disclosed in US2O II 0065902A I or is (or comprises) such antibody or a PCSK9-binding derivative thereof [007121 In an embodiment, the ligand is or comprises the variable domains of antibody alirocumab or SAR236553/REGN727 (Sanofi Aventis/Regeneron) or is (or comprises) such antibody or a PCSK9-binding derivative thereof In an example, the alirocumab is gycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos or Hek293 cell).

Preferably, the ligand is alirocumab or SAR2365531REGN727.

[007131 In an embodiment, the ligand is or comprises the variable domains of antibody evolocumab or or is (or comprises) such antibody or a PCSK9-binding derivative thereof In an example, the antibody is glycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos or Hek293 cell). Preferably, the ligand is evolocumab.

1007141 In an embodiment, the ligand is selected from evolocumab, I D05-lgG2 (Merck & Co.), ALN-PC S02 (Alnylam), RN3 16 Pfizer-Rinat) and alirocumab.

[007151 In all embodiment, the ligand is selected from the following (sequences and definitions as per US2OI 1/0065902, incorporated herein by reference):- 1, An antibody or antigen-binding fragment thereof which specifically binds hPCSK9, wherein the antibody or antigen-binding fragment comprises the heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pair having SEQ ID NOs: 218/226.

2, The antibody or antigen-binding fragment of concept comprising heavy and light chain CDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.

3. The antibody or antigen-binding fragment of concept 2 comprising an HCVR having the amino acid sequence of SEQ ID NO: 218 and an LCVR having the amino acid sequence of SEQ ID NO: 226.

4. An antibody or antigen-binding fragment thereof which binds to the same epitope on hIPCSK9 as an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.

5. An antibody or antigen-binding fragment thereof which competes for binding to hPCSK9 with an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.

[007161 hI an embodiment, the ligand is selected from the following (sequences and definitions as per US2012/0093818, incorporated herein by reference):- 1. An isolated neutralizing antigen binding protein that binds to a PCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1, wherein the neutralizing antigen binding protein decreases the LDLR lowering effect of PCSK9 on LDLR, wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60.

2. The isolated neutralizing antigen binding protein of concept 2, wherein the antigen binding protein is a LDLR non-competitive neutralizing antigen binding protein.

3. The isolated neutralizing antigen binding protein of concept 2, wherein the antigen binding protein is a LDLR competitive neutralizing antigen binding protein.

4. An antigen binding protein that selectively binds to PCSK9, wherein said antigen binding protein binds toPCSK9 with aKd that is less than 100 pM.

5. An antigen binding protein that binds to a PC SIC 9 protein of SEQ ID NO: 303 in a first manner, wherein the antigen binding protein binds to a variant of PCSK9 in a second manner, wherein said PCSK9 variant has at least one point mutation at a position selected from the oup consisting of 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, t2c,3fl, 313, 337, sic, 521, and 554 of SEQ ID NO: 303, wherein the first manner comprises a first EC5O, a first Bmax, or a first EC5O and a first Bmax, wherein the second manner comprises a second EC5O, a second Bmax, or a second EC5O and a second Bmax, and wherein a value for the first manner is different from a value for the second manner, and wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60.

6. The antigen binding protein of concept 6, wherein the first manner comprises a first Bmax, wherein the second manner comprises a second Bmax that is different from the first Bmax, and wherein said PCSK9 variant has at least one point mutation selected from the group consisting of: D162R, R164E, E167R, S]23R, E]29R, A3LIR, D313R, D337R, R519E, HS2IR, and QSS4R.

7. The antigen binding protein of concept 6, wherein the antigen binding protein binds to PCSK9 at a location that overlaps with a location that LDLR binds to PCSK9, 8, A method of making an antigen binding protein that binds to a PCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1, wherein the antigen binding protein decreases the LDLR lowering effect ofPCSK9 on LDLR, said method comprising:providing a host cell comprising a nucleic acid sequence that encodes the antigen binding protein; andmaintaining the host cell under conditions in which the antigen binding protein is expressed, wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60.

9. A method for treating or preventing a condition associated with elevated serum cholesterol levels in a subject, said method comprising administering to a subject in need thereof an effective amount of an isolated neutralizing antigen binding protein simultaneously or sequentially with an agent that elevates the availability of LDLR protein, wherein the isolated antigen binding protein binds to a PCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1, wherein the neutralizing antigen binding protein decreases the LDLR lowering effect of PCSK9 on LDLR, wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60.

10. The method of concept 10, wherein the agent that elevates the availability of LDLR protein comprises a statin.

11. An antigen binding protein that binds to PCSK9, wherein when the antigen binding protein is bound to PCSK9, the antibody is positioned 8 angstroms or less from at least one of the following residues ofPCSK9: 5153, 5188, 1189, Q190, 5191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, 5376, 1377, F379, 1154, T187, 1-1193, E195, 1196, M201, V202, C223, 1228, 5235, G236, A239, G244, M247, 1369, S372, C375, or C378, wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60.

1007171 The ligand call be used for the treatment, therapy, prophylaxis and/or diagnosis of one or more diseases or conditions or susceptibility thereto, wherein such diseases or conditions comprise those disclosed in US2O 2009381 8A I (Amgen, mc) and TJS2O1 10065902A1 (Regeneron Pharmaceuticals, mc), eg, a disease or condition disclosed in paragraphs [0375] to [0383] ofUS20120093818A1, which disclosure is incorporated herein by reference in its entirety for inclusion in one more claims herein.

1007181 The ligand can be administered to a human characterised as described in US20120093818A1 (Amgen, mc) orUS2Ol 10065902A1.

1007191 The ligand carl be administered in a form or combination disclosed in U520120093818A1 (Amgen, mc) or U5201 10065902A1, which disclosure is incorporated herein by reference. For example, the ligand with a drug, excipient, diluent or carrier as described in US201200938 1 8A1 (Amgen, mc) or US2O1 I 0065902A1 (eg, as disclose in paragraphs [0384] to [0412] ofUS20I20093818A1), which disclosure is incorporated herein by reference, and the present invention also relates to the corresponding pharmaceutical compositions comprising the combination of a ligand of the invention and such a further agent.

1007201 The ligand can be used in a method of diagnosis as set out in US2OI 2009381 8A I (Amgen, mc) or US2O 11 0065902A1, eg, in paragraphs [0413] to [0415] of US20120093818A1 which disclosure is incorporated herein by reference.

1007211 Diagnostic Applications 1007221 In some embodiments, the ligand of the invention is a diagnostic tool, The ligand can be used to assay the amount of PCSK9 present in a sample and/or subject. As will be appreciated by one of skill in the art, such ligands need not be neutralizing ligands. In some embodiments, the diagnostic ligand is not a neutralizing ligand. In some embodiments, the diagnostic ligand binds to a different epitope than a neutralizing ligand binds to. In some embodiments, the two ligands do not compete with one another.

[007231 In some embodiments, the ligands of the invention are used or provided in an assay kit and/or method for the detection of PCSK9 in mammalian tissues or cells in order to screen/diagnose for a disease or disorder associated with changes in levels of PCSK9. The kit comprises a ligand that binds PCSK9 and means for indicating the binding of the ligand with PCSK9, if present, and optionally PCSK9 protein levels. Various means for indicating the presence of a ligand can be used. For example, fluorophores, other molecular probes, or enzymes can be linked to the ligand and the presence of the ligand can be observed in a variety of ways. The method for screening for such disorders can inv6lve the use of the kit, or simply the use of one of the disclosed ligands and the determination of whether the ligand binds to PCSK9 in a sample. As will be appreciated by one of skill in the art, high or elevated levels of PCSK9 will result in larger amounts of the ligand binding to PCSK9 in the sample.

Thus, degree of ligand binding can be used to determine how much PCSK9 is in a sample.

Subjects or samples with an amount ofPCSK9 that is greater than a predetermined amount (e.g., an amount or range that a person without a PCSK9 related disorder would have) can be characterized as having a PCSK9 mediated disorder, In some embodiments, the invention provides a method wherein the ligand is administered to a subject taking a statin, in order to determine if the statin has increased the amount of PCSK9 in the subject.

[007241 In some embodiments, the ligand is a non-neutralizing ligand and is used to determine the amount of PC SK9 in a subject receiving an ABP and/or statin treatment.

[007251 lii some embodiments, the ligand of the invention can specifically bind human PCSK9 (eg, one, two or more rare variant forms disclosed herein) and is characterized by at least one of: (i) capable of reducing serum total cholesterol at least about 25-35% and sustaining the reduction over at least a 24 day period relative to a predose level; (ii) capable of reducing serum LDL cholesterol at least about 65-80% and sustaining the reduction over at least a 24 day period relative to a predose level; (iii) capable of reducing serum [DL cholesterol at least about 40-70% and sustaining the reduction over at least a 60 or 90 day period relative to a predose level; (iv) capable of reducing serum triglyceride at least about 25-40?.'b relative to predose level; (v) does not reduce serum FIDL cholesterol or reduces serum MDL cholesterol no more than 5?/ relative to predose level. In some embodiments, an isolated nucleic acid molecule is provided and it encodes the ligand. In some embodiments an expression vector is provided and comprises the nucleic acid molecule. In some embodiments, a pharmaceutical composition is provided and it can comprise the ligand and a pharmaceutically acceptable carrier. In some embodiments, a method is provided for treating a disease or condition which is ameliorated, improved, inhibited or prevented with a PCSK9 antagonist ligand of the invention. The method can comprise administering a therapeutic amount of the pharmaceutical composition or ligand to a subject in need thereof In some embodiments, the subject is a human subject suffering from hypercholesterolemia, hyperlipidemia, indicated for [DL apheresis, identified as heterozygous for Familial Hypercholesterolemia, statin intolerant. statin uncontrolled, at risk for developing hypercholesterol emi a, dyslipi demi a, chol estati c liver disease, nephroti c syndrome, hypothyroidism, obesity, atherosclerosis and cardiovascular diseases. In some embodiments, a method of providing a treatment or therapy is provided to a subject. In some embodiments, the method comprises reducing serum cholesterol at least about 40-70% over at least 60 to 90 days. In some embodiments, a method of receiving treatment or therapy is provided, the method can comprise receiving a ligand thereof at a frequency of once every 60 to 90 days.

[007261 In one aspect, the invention provides a ligand of the invention which is or comprises an human antibody or antigen-binding fragment of a human antibody that specifically binds and inhibits human proprotein convertase subtilisin/kexin type 9 (hPCSK9, eg, one, two or more rare variant forms disclosed herein and optionally form a and/or form a'), characterized by the ability to reduce serum LDL cholesterol in a human by 40-80% over a 24, 60 or 90 day period relative to predose levels, with little or no reduction in serum HDL cholesterol and/or with little or no measurable effect on liver function, as determined by ALT and AST measurements.

[007271 lii one embodiment, the ligand of the invention comprises an antibody or antigen-binding fragment of an antibody that specifically binds hPC SK9 and is characterized by at least one of: (i) capable of reducing serum total cholesterol at least about 25-35% and sustaining the reduction over at least a 24 day period relative to a predose level, preferably the reduction in serum total cholesterol is at least about 30-40%; (ii) capable of reducing serum LDL cholesterol at least about 65-80% and sustaining the reduction over at least a 24 day period relative to a predose level; (Hi) capable of reducing serum triglyceride at least about 25-40?.' relative to predose level; (iv) does not reduce serum MDL cholesterol or reduces serum HDL cholesterol no more than 5% relative to predose level.

[007281 See US2O1 1/0065902 for definitions of these terms and optional features, the disclosure of which is incorporated herein by reference in its entirety.

1007291 In one embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody that specifically binds hPCSK9 and is characterized by at least one of: (i) capable of reducing serum LDL cholesterol at least about 40-70% and sustaining the reduction over at least a 60 or 90 day period relative to a predose level; (ii) capable of reducing serum triglyceride at least about 25-40% relative to predose level; (iii) does not reduce serum HDL cholesterol or reduces serum HDL cholesterol no more than 5% relative to predose level.

1007301 In one embodiment, the antibody or antigen-binding fragment is characterized as exhibiting an enhanced binding affinity (1(D) for hPCSK9 at pH 5.5 relative to the lCD at pH 7,4, as measured by plasmon surface resonance. In a specific embodiment, the antibody or fragment thereof exhibits at least a 20-fold, at least a 40-fold or at least a 50-fold enhanced affinity for PCSK9 at an acidic pH relative to a neutral pH, as measured by surface plasmon resonance.

1007311 In one embodiment, the antibody or antigen-binding fragment is characterized as not exhibiting an enhanced binding affinity for PCSK9 at an acidic pH relative to a neutral pH, as measured by surface plasmon resonance, In a specific embodiment, the antibody or fragment thereof exhibits a decreased binding affinity at an acidic pH.

1007321 lii another embodiment, the antibody or antigen-binding fragment binds human, human GOP mutation D374Y, cynomolgus monkey, rhesus monkey, mouse, rat and hamster PC S 1(9.

1007331 lii one embodiment, the antibody or antigen-binding fragment binds human and monkey PCSK9, but does not bind mouse, rat or hamster PCSK9.

1007341 In one embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody comprising one or more of a heavy chain variable region (HCVR), light chain variable region (LCVR), HCDR1, HCDR2, HCDR3 disclosed in any of paragraphs [023]-[037] of IJS2OI 1/0065902, the disclosures of which are incorporated herein by reference.

1007351 In a related embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody which specifically binds hPCSK9, wherein the antibody or fragment comprises heavy and light chain CDR domains contained within heavy and light chain sequence pairs selected from the group consisting of SEQ ID NO (using the sequence numbering in U52011/0065902): 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 1t4/116, 1t8/t20, t22/130, t38/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264, 266/274, 282/284, 286/288, 290/298, 306/308, 310/3 12, 3 14/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384, 386/394, 402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452, 454/456, 458/466, 474/476, 478/480, 482/490, 498/500, 502/504, 506/5 14, 522/524, 526/528, 530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586, 594/596, 598/600, 602/6 10, 618/620, 622/624, 626/634, 642/644, 646/648, 650/658, 666/668, 670/672, 674/682, 690/692, 694/696, 698/706, 714/716, 718/720, 722/730, 738/740 and 742/744. In one embodiment, the CDR sequences are contained within HCVR and LCVR selected from the amino acid sequence pairs of SEQ ID NO: 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 122/130, 138/140, 142/144, 218/226, 234/236, 23 8/240, 242/250, 258/260, 262/264, 3 14/322, 330/332 and 334/336. k more specific embodiments, the CDR sequences are comprised within HCVR/LCVR sequences selected from SEQ ID NO: 90/92 or 218/226, [007361 In an example, the invention features a pharmaceutical composition comprising a ligand of the invention, wherein the ligand comprises or consists of a recombinant human antibody or fragment thereof which specifically binds hPCSK9 and a pharmaceutically acceptable carrier. In one embodiment, the invention features a composition which is a combination of a ligand of the invention (eg, an antibody or antigen-binding fragment of an antibody), and a second therapeutic agent. The second therapeutic agent may be any agent that is advantageously combined with the ligand of the invention, for example, an agent capable of inducing a cellular depletion of cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl (HIvIG)-coenzyme A (C0A) reductase, such as, for example, cerovastatin, atorvastatin, simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc; capable of inhibiting cholesterol uptake and or bile acid re-absorption; capable of increasing lipoprotein catabolism (such as niacin); and/or activators of the LXR transcription factor that plays a role in cholesterol elimination such as 22-hydroxycholesterol.

[007371 In an example, the invention provides a method for inhibiting hPCSK9 activity using the anti-PC 51(9 ligand of the invention (eg, an antibody or antigen-binding portion of the antibody of the invention), wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody of the invention, The disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition or reduction of PCSK9 activity. Specific populations treatable by the therapeutic methods of the invention include subjects indicated for LDL apheresis, subjects with PC SK9-activating mutations (gain of function mutations, "GOF"), subjects with heterozygous Familial Hypercholesterolemia (heFH; subjects with primary hypercholesterolemia who are statin intolerant or statin uncontrolled; and subjects at risk for developing hypercholesterolemia who may be preventably treated. Other indications include dyslipidemia associated with secondary causes such as Type 2 diabetes mellitus, cholestatic liver diseases (primary biliary cirrhosis), nephrotic syndrome, hypothyroidism, obesity; and the prevention and treatment of atherosclerosis and cardiovascular diseases.

1007381 In specific embodiments of the method of the invention, the ligand of the invention (eg, anti-hPCSK9 antibody or antibody fragment of the invention) is useful to reduce elevated total cholesterol, non-HDL cho'esterol, LDL cholesterol, andlor apolipoprotein B (apolipoprotein B 100).

1007391 The ligand (eg, antibody or antigen-binding fragment) of the invention may be used alone or in combination with a second agent, for example, an HMG-CoA reductase inhibitor and/or another lipid lowering drug.

[007401 Jeatnient J)0J)7(1(Jt707 [007411 The invention provides therapeutic methods for treating a human patient in need of a composition orligand of the invention. While modifications in lifestyle and conventional drug treatment are often successful in reducing cholesterol levels, not all patients are able to achieve the recommended target cholesterol levels with such approaches. Various conditions, such as familial hypercholesterolemia (FM), appear to be resistant to lowering of LDL-C levels in spite of aggressive use of conventional therapy. Homozygous and heterozygous familial hypercholesterolemia (hoFH, heFH) is a condition associated with premature atherosclerotic vascular disease, However, patients diagnosed with hoFH are largely unresponsive to conventional drug therapy and have limited treatment options. Specifically, treatment with statins, which reduce LDL-C by inhibiting cholesterol synthesis and upregulating the hepatic LDL receptor, may have little effect in patients whose LDL receptors are non-existent or defective. A mean LDL-C reduction of only less than about 20?/ has been recently reported in patients with genotype-confirmed hoFH treated with the maximal dose of statins. The addition of ezetimibe 10 mg/day to this regimen resulted in a total reduction of LDL-C levels of 27%, which is still far from optimal. Likewise, many patients are statin non-responsive, poorly controlled with statin therapy, or cannot tolerate statin therapy; in general, these patients are unable to achieve cholesterol control with alternative treatments. There is a large unmet medical need for new treatments that can address the short-comings of current treatment options, 1007421 Specific populations treatable by the therapeutic methods of the invention include patients indicated for LDL apheresis, subjects with PCSK9-activating (GOF) mutations, heterozygous Familial Hypercholesterolemia (heFH); subjects with primary hypercholesterolemia who are statin intolerant or statin uncontrolled; and subjects at risk for developing hypercholesterolemia who may be preventably treated, 1007431 Therapeutic Administration and Formulations 1007441 The invention provides therapeutic compositions comprising the anti-PCSK9 ligands, antibodies or antigen-binding fragments thereof of the present invention. The administration of therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa, These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTTM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (t998) JPharm Sci Technol 52:238-3 ii.

1007451 The dose may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When the ligand, eg, antibody, of the present invention is used for treating various conditions and diseases associated with PCSK9, including hypercholesterolemia, disorders associated with LDL and apolipoprotein B, and lipid metabolism disorders, and the like, in an adult patient, it is advantageous to intravenously administer the ligand or antibody of the present invention normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight.

Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted.

1007461 Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, thus the composition invention provides the ligand by e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol, Chem, 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local, 1007471 The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid,), 1007481 In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefion (1987) CRC Crit, Ref Biomed, Eng, 14:201), In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres,, Boca Raton, Fla. (1974), In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e,g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984), 1007491 The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known, For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e,g,, ethanol), a polyalcohol (e,g,, propylene glycol, polyethylene glycol), a nonionic surfactant [eg., polysorbate 80, 1-ICO-SO (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule. A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention, Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge, Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device.

Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[007501 Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention, Examples include, but certainly are not limited to AUTOPENTM (Owen Mumford, Inc., Woodstoclc, UK), DISETRONICTM pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUIvIALOG MIX 75/25TM pen, HUI[VIALOGTM pen, HUIIVIALIIN 70/3OTM pen (Eli Lilly and Co., Indianapolis, md.), NOVOPENTMI, II and lU (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes, NJ.), OPTIPENTTM, OPTIPEN PROTM, OPTIPEN STARLETTM, and OPTICLIKTTM (sanofi-aventis, Frankfurt, Germany), to name only a few, Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTARTM pen (sanofi-aventis), the FLEXPENTM (Novo Nordisk), and the KWIKPENTM (Eli Lilly).

[007511 Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100mg and in about 10 to about 250 mg for the other dosage forms.

[007521 The invention provides therapeutic methods in which the ligand, eg, antibody or antibody fragment, of the invention is useful to treat hypercholesterolemia associated with a variety of conditions involving hPCSK9. The anti-PCSK9 ligands, eg, antibodies or antibody fragments, of the invention are particularly useful for the treatment of hypercholesterolemia and the like, Combination therapies may include the anti-PC SK9 ligand of the invention with, for example, one or more of any agent that (I) induces a cellular depletion of cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase, such as cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin; (2) inhibits cholesterol uptake and or bile acid re-absorption; (3) increase lipoprotein catabolism (such as niacin); and activators of the LXR transcription factor that plays a role in cholesterol elimination such as 22-hydroxycholesterol or fixed combinations such as ezetimibe plus simvastatin; a statin with a bile resin (e.g., cholestyramine, colestipol, colesevelam), a fixed combination of niacin plus a statin (e.g., niacin with lovastatin); or with other lipid lowering agents such as omega-3-fatty acid ethyl esters (for example, omacor).

1007531 Tailoring Antibodies to Rare PCSK9 Variant Profile 1007541 The invention includes the possibility to tailor treatment of humans frirther by selecting antibody-based ligands with variable domains based on gene segments commonly found in humans of the ethnic populations where the variant PCSK9 forms are found to meet the selection criteria of the invention. An example is provided below for ligands comprising antibody VH domains derived from recombination of human VH3-23, 1007551 The inventor analysed the frequencies and distribution of various human VF-13-23 alleles and realised the desirability of using ligands based on human VH3-23 alleles comprising SNP rs56069819. This SNP corresponds to a change from leucine at position 24 in the encoded protein sequence to a valine at that position (L24V change) and the SNP is at coordinate 106725482 on human chromosome 14.

1007561 Figure 2 shows the cumulative allele frequency distribution across the 1000 Genomes Project database of human VH3-23 alleles comprising SNP rs560698i9 (such alleles denonted "C" and the most frequent allele (which does not comprise this SNP) denoted "A"). The figure shows that VH3-23 alleles comprising SINP rs56069819 are present at a cumulative frequency of 11% across all human ethnic populations taken as a whole, whereas in certain specific human ethnic sub-populations (ASW, LWK, YRI, CEU and GBR) such alleles are present at an above-average cumulative frequency. Indicated in the figure are those human PCSK9 variant forms (marked "Variants") that are found in the various sub-populations with above-average occurrence of human VH3-23 alleles comprising SNP rs360698 19. Table 6 shows the VI-13-23 variants and the SNPs that they comprise, as well as their cumulative allele frequencies as found in the 1000 Genomes Project database.

1007571 Notably, human V113-23 alleles comprising SNP rs56069819 were found in the CEU population at a frequency that is almost double the frequency of 11% for all populations. For the ASW and YRI populations the frequency was over a quarter of the population. Thus, the invention advantageously enables one to select a ligand comprising an antibody or antibody fragment, wherein the antibody or fragment comprises a VH domain derived from the recombination of a human VH gene segment, a human D gene segment and a human iii gene segment, the VH gene segment comprising a nucleotide sequence that comprises SNP rs56069819 (dbSNP numbering, build number as recited above).

[00758J In an example, one can tailor the treatment further by selecting such a ligand that specifically binds to a human PCSK9 selected from forms:f c, m, e h p, q and a], such forms being those appearing in human populations ASW, LWK, YRI, CEU and GBR.

[00759J In an example, the Vii gene segment is VH323*O4, which is a commonly found variant that comprises SNP rs56069819 in human populations ASW, LWK, YRJ CEU and (3BR.

[00760J In an example, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human that expresses a human PCSK9 selected from forms:f c, m, e, Ii, p. q and af.

1007611 in an example, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human of ASW, LWK, YRI, CEU or GBR ancestry.

1007621 In an embodiment, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human of ASW ancestry, wherein the human expresses a PCSK9 selected fromf c in, e, h p and q or the human comprises a corresponding nucleotide or amino acid sequence as set out in TableS. Optionally this ligand comprises a VH domain derived from recombination of human VH323*O4.

1007631 In an embodiment, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human of LWK ancestry, wherein the human expresses a PCSK9 selected fromf c, in, e and h or the human comprises a corresponding nucleotide or amino acid sequence as set out in Table 5. Optionally this ligand comprises a Vii domain derived from recombination of human VH323*O4.

1007641 In an embodiment, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human of YR1 ancestiy, wherein the human expresses a PCSK9 selected fromf c, in, e and h or the human comprises a corresponding nucleolide or amino acid sequence as set out in Table 5. Optionally this ligand comprises a VH domain derived from recombination of human VH323*O4.

[007651 In an embodiment, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human of CETJ ancestry, wherein the human expresses a PCSK9 selected fromf c, p and cii or the human comprises a corresponding nucleotide or amino acid sequence as set out in Table 5. Optionally this ligand comprises a VII domain derived from recombination of human VH3 23*34 1007661 In an embodiment, the ligand is for treating and/or preventing a PCSK9-mediated disease or condition in a human of GBR ancestry, wherein the human expresses a PCSK9 selected fromf c andp or the human comprises a corresponding nucleotide or amino acid sequence as set out in Table 5. Optionally this ligand comprises a VII domain derived from recombination of human VFI3-23 *Q4 1007671 In an example, the ligand is alirocumab.

1007681 References: IIorton eta!, Trends Biochem Sci. 2007 Feb;32(2):71-7, Epub 2007 Jan), Molecular biology ofPCSK9: its role in LDL metabolism.

Seidah and Prat, J Mol Med (BerI). 2007 JuI;85(7):685-96. Epub 2007 Mar 10, The proprotein convertases are potential targets in the treatment of dyslipidemia.

Benjannet et at, J Biol Chem. 2004 Nov 19;279(47):48865-75. Epub 2004 Sep 9, NARC- 1!PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.

Lagace eta!, J Clin Invest. 2006 Nov; 116(1 1):2995-3005, Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.

Maxwell eta!, Proc NatI Acad Sci U S A. 2005 Feb 8;]02(6):2069-74, Epub 2005 Jan 27, Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment.

Park eta!, J Biol Chem. 2004 Nov 26;279(48):50630-8, Epub 2004 Sep 22, Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver.

Rashid ci cii, Proc Natl Acad Sci USA. 2005 Apr 12;102(15):5374-9, Epub 2005 Apr 1, Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9, Kotowski ci at, Am J Hum Genet. 2006 Mar;78(3):410-22. Epub 2006 Jan 20, A spectmm of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol.

Chen eta!, JAm Coil Cardiol, 2005 May 17;450):l6lI-9, Epub 2005 Apr2t, A common PCSK9 haplotype, encompassing the E670G coding single nucleotide polymorphism, is a novel genetic marker for plasma low-density lipoprotein cholesterol levels and severity of coronary atherosclerosis.

Pisciotta etal, Atherosclerosis. 2006 Jun;186(2):433-40, Epub 2005 Sep 23, Additive effect of mutations in LDLR and PCSK9 genes on the phenotype of familial hypercholesterolemia.

Zhao eta!, Am J Hum Genet. 2006 Sep;79(3):5 14-23. Epub 2006 Jul 18, Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote, Seidah etal, Proc Nati Acad Sci U S A. 2003 Feb 4;]00(3):928-33. Epub 2003 Jan 27, The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.

Table 3: 1000 GENOMES PROJECT IRflfl\ POPULATIONS BeIo is a summar\ ol 1110 ethnic populations as per the I Genonies ProleeL sequences Population European aucestr Itah residents (([P11) with Northern and Western [uropean ancestn (( LI) Toscaiii iii II;tl Ia (TSI liii Ii sh lioni LnuI and and Scoiland I GBR Finni sli 110111 Fi IlIflIid (FIN) lbei ian populations ii Spin (IRS) East Asian ancestr I Ian Chinese in Ueijin.. China (C 1W Japanese in To ko. Japan (JPT) I Ian Chinese South (CI IS) (hi nese ill \isIlLlitIlLtbaIlIla ((DX Kinh in I In (hi \Iinh C itv, Vietnam C CI IV) Chinese in Denver. colorado (I ID) (pilot 3 unIv \% esi African anceslrv ori.i ha in I bad a a. Ni ueri a ( V RI) I uhva iii \VehLI\ e. KeIl\ 1 (I.\\ K) C rambian in \\e'tei-n Di si on. The C iamhia (C 1WD \ IaIav ian in BInk re \Ialawi ( \I LB) West African Population (TBD) A iiiericas All jean Aiicesti s in Southv,L'st VS C ASW) \I'iicaii \me1e1tn iii Jackson, MS C \JM Ati-ican Caribbean iii Barbados (AC B) Population \lexican.\ncestr in I Os \imelcs. (A CMXL) Puerto Rican in Puerto Rico (Pt'R) (oInnihial1 in \Iedellin. (olonihia (CI NJ) l'enl\-i Ill in Linia Peru (PEL Sou iii.%sian a riced rs Alit_mi iii the Slate ol' *\ssam, lath a Ka adiha in (alcLuta, India Redd in 11' derahad lad ia \ laratha in l3omha India Pu nj at) i in I a h ore. Pa hi stan Table 4: TOIs & Related Diseases, Conditions and Example Ligands Human TOl Example Disease or Example Ligand Condition fl-I 7a Inflammatory Disease A11N457 Lveitis Ixekizumab Rheumatoid Arthritis Psoriasis Angiotensin 11 Receptor Type 1 (AT1) Hypertension LCZ696 Neprilysin Hypertension LCZ696 Metabotropic Glutamate Receptor 5 (Mghir5) Fragile X Syndrome AFQOS6 Mavoglurant A Histone Deacetylase Cancer, Eg, Multiple LBHSS9 Myeloma Panobinostat Diacyiglycerol acyltransferase-1 (DGAT-1) Familial LCQ9OS Chylomicronaemia Pradigastat Sdrome Smoothened (Snio) Basal cell carcinoma LDE225 Solid tumour Myelofi b rosi s Medulloblastoma Solid tumour Smoothened (Snio) receptor Basal cell carcinoma LEQSO6 Solid tumors ALK Non small cell lung LDK37S carcinoma (NSCLC) phosphatidylinositol-3-kinase (P13K) Cancer. eg. solid tumour. BKM12O mTOR breast cancer, Renal cell AKT carcinoma (RCC), Endometrial cancer, Non-small cell ung cancer (NSCLC). prostate cancer.

Glioblastoma multiforme (GBM)

CRPC

GIST

Myelofibrosis mTOR Cancer. eg, solid tumour, BEZ235 P13K breast cancer, Renal cell AKT carcinoma (RCC), Endomctrial cancer Non-small cell lung cancer (NSCLC) prostate cancer.

GlioMastoma multiforme (GBM)

CRPC

GIST

Myelofibrosis PI3Ka Canccr. eg, solid tumour. BYL719 mTOR breast cancer, Renal cell P13K carcinoma (RCC), AKT Endornetrial cancer, Non-small cell ung cancer (NSCLC). prostatc cancer.

Glioblastoma multifonne (GBM)

CRPC

GIST

Myelofibrosis Mitogen-activated ERIC kinase 1 (MEKI) Cancer. eg. solid tumour. MEKIG2 Mitogen-activated ERIC kinase 2 (MEK2) melanoma, pancreatic, colon, lung or thyroid canccr Metastasis protein kinase Acute myeloid leukemia PKC4I2 protein kinase C (AML) Midostaurin FLT-3 Myelodvsplastic syndrome c-ICIT (MDS) Aggressive systemic rnastocvtosi s (ASM) ActRIIB Sporadic inclusion body BYM338 myositis (sIBM) bimagrumab CD19 * Cancer. eg. CTLO19 (formerly CART-Leukaemia, mast 19) cell leukemia, Acute Lymphoblastic Leukemia Chronic lymphocytic leukemia (CLL) or Hematological tumors II -hydroxyase Cushing's syndrome LC1699 BRAF Cancer. eg. melanoma LGX8 IS Receptor Tyrosine Kinase * Cancer. eg. solid TK1258 (formerly CI-JIR-FGFR tumour 258) * Breast Cancer Dovitinib * Endometrial cancer * Hepatocellular carcinoma * Renal cell carcinoma (RCC) * Bladder cancer * multiple myeloma (MM), hepatocellular carcinoma * endometrial cancer DAC enzyme hematologic malignancy LBH5S9 Multiple mveloma panobinostat Myel\odysplastic syndrome (MDS) Myelofi brosi s HSP9O * Cancer. eg, Breast AUY922 Cancer. gastric cancer or Non-small cell lung cancer NSCLC) FGFR * Cancer. eg. solid BGJ 398 tumour * Breast Cancer * Endometrial cancer * Hepatoceflular carcinoma * Renal cell carcinoma (RCC) * Bladder cancer * multiple myeloma (MM), hepatocelluhir carcinoma * cndometrial cancer cIAP1 * Cancer. cg. solid LCLI61 cIAP2 tumour * Breast Cancer * Endometrial cancer * Hepatocellular carcinoma * Renal cell carcinoma (RCC) * Bladder cancer * muitiple rnvelonia (MM), hepatocelluhir carcinoma * endometrial cancer Akt * CanceL eg, Solid GDC-0068 tumour, gastric RG7440 cancer (eg, castration-resistant prostate cancer), prostate cancer gastroesophage& junction cancer CD22 Hematologic malignancies DCDT29SOS Non-Hodgkin's lymphoma RG75 93 (eg, relapsed or refractory follicular non-Hodgkin's vniphoina) Diffuse large B-cell lymphoma (eg, relapsed or refractory diffuse arge B-cell lyniphoma) CD79b Hematologic malignancies DCDS45O1A Non-Hodgkin's lymphoma RG7596 (eg, relapsed or refractory follicdar non-Hodgkin's Nmphoma) Diffuse large B-cell lymphoma (eg, relapsed or refractory diffuse arge B-cell lymphoma) endothelin B receptor (ETBR) DEDN6526A Melanoma. eg. metastauc RG7636 or unresectabk mdanoma HER3 Cancer. eg, metastatic MEFID7945A epitheli& tumour. RG7597 metastatic squamous cell carcinoma of the head and neck cancer. metastatic colorectal cancer EGFR Cancer. eg, metastatic MEFID794SA epitheli& tumour. RG7597 metastatic squamous cell Necitumumab carcinoma of the head and neck cancer. metastatic colorectal cancer MUC16 Cancer. cg. ovarian cancer DMUC5754A (eg, pkitinurn-resistant RG745 8 ovarian cancer) sodium-dependent phosphate transport protein Cancer. eg. metastatic 11011-DNTBO600A 2b (NaPi2b) squamous non-small cell RG7599 lung cancer, ovarian cancer (eg, platinum-resistant ovarian cancer) PDL I (programmed death ligand I) Cancer. eg. solid tumour MPDL328OA metastatic melanoma RG7446 non-small cell llLng cancer STEAP1 (six-transmcmbranc cpithclial Cancer. cg, prostate cancer DSTP3OS6S antigen of the prostate 1) (eg, metastatic castration-R07450 resistant prostate cancer) BcI-2 Cancer. eg. leukaemia (eg. GDC-O1 99 chronic lymphocvtic RG760 1 lcukaemia) non-Hodgkin's lymphoma Checkpoint kinase 1 (ChK1) GDC-0425 Cancer. eg, solid tumouL R07602 refractory solid tumour or lvmphoma GDC-0575 RG7741 mitogcn activated protein kinasc kinasc GDC-0973 (MAPKK) R0742 1 Cancer. eg, solid tumour.

Cobimetinib melanoma (cg. mctastatic

MEK

melanoma) GDC-0623 RG7420 cpidcrmal growth factor donmin-likc-7 Cancer. cg, colorcctal Parsatuzumab (EGFL7) cancer (eg, metastatic MEGFO444A colorectal cancer). NSCLC RG7414 phosphatidylinositol-3-kinase (P13K) Cancer. eg. prostate cancer, GDC-0032 renal cell carcinoma. RG7604 endonietrial cancer, breast cancer (eg, HER2-negative GDC-0084 metastatic breast cancer, R07666 metastatic hornione receptor-positive breast Pictilisib cancer), solid tumour GDC-094 1 R07321 mTOR Cancer. eg. prostate cancer. GDC-0980 TORCI renal cell carcinoma. RG7422 TORC2 endonietrial cancer, breast P13K cancer (eg, HER2-negative metastatic breast cancer, nietastatic hormone receptor-positive breast cancer), solid tumour 1L17 Autoimmune disease Secukinumab Ankylosing spondylitis; ixekizumab Asthma; Multiple myeloma; Multiple sclerosis; Polymyalgia rheumatica; Psoriasis; Psoriatic arthritis; Rheumatoid arthritis; Lveitis Ml prime segment of membrane IgE Quilizumab Allergic Asthma MEMP1972A RG7449 TFN alpha Autoimmune disease Rontalizumab Systemic lupus ervthematosus PCSK9 (proprotein convertase subtilisinlkexin coronary heart disease MPSK3169A type 9) (CHD) or high risk of CHD RG7652 hyperlipidaemia liyperch ole ster& aemi a stroke atherosclerosis a cardiovascular disease or condition a condition associated with elevated LDL Vascu'ar Endothelial Growth Factor-A Diabetic Macular Edema EYLEA® (VEGF-A) (DME). Aflibercept Branch Retinal Vein AVASTIN'TM Placental Growth Factor (P1GF) Occlusion (BRVO) LUCENTISThI Wet age-related macular degeneration (Wet AMD) IL-6 receptor lnflammatoi disease. eg, Sarilumab rheumatoid arthritis REGN88 Uveitis (eg, non-infectious uveitis) Ankylosing spondylitis; Canccr PCSK9 (proprotein convertase subtilisinlkexin coronary heart disease Alirocumab type 9) (CHD) or high risk of CHD RE6N727 hvperlipidaemia LY30 15014 livpercholesteroaernia stroke atherosclerosis a cardiovascular disease or condition a condition associated vith elevated LDL NGF Osteoarthritis Fasinumab Pain REGN475 TL-4 receptor alpha Allergic asthma Dupilumab eosinophilic asthma REGN66S IL-13 receptor Atopic dermatitis delta-like ligand-4 (D114) Enoticumab Cancer REGN421 angiopoietin-2 (Ang2) Ncsvacumab Cancer REGN91O GDF8 Mctabolic disorder REGNIO33 cancer, obesity, diabetes, LY2495655 arthritis, multiple sclerosis.

musell]ar dystrophy, aniyotrophic lateral sclerosis, Parkinson's disease, osteoporosis, osteoarthnfls. osteopenia.

metabolic syndromes (including. but not limited to diabetes, obesity, nutritional disorders, organ atrophy, chronic obstructive pulmonary disease and anorexia) Disuse niusck atrophy cancer-related cachexia ERBB3 Cancer REGNI400 angiopoietin -1 (AngI) AMG386 Cancer, eg, ovarian cancer angiopoictin -1 (Ang2) VEGF receptor Cancer Motesanib PDGF receptor tSCLC AMG 706 Stem cefl factor receptor Breast cancer Thyroid cancer type 1 insulin-like growth factor receptor Cancer Ganitumab (IGF1R) Breast cancer Linsitinib Pancreatic cancer ASP7487 hepatocyte growth factor (HGF) Cancer Riloturnumab Breast cancer Pancreatic cancer HER3 Cancer AMG 888 ErbB3 Breast cancer U3-1 287 Pancreatic cancer IL-17 receptor Inflammator disease AMG 827 Asthma brodalumab Psoriasis sclerostin Bone-related condition AMG 785 postmenopausal CDP7S5 1 osteoporosis fracture healing glucokinase AMG 151 Diabetes ARRY-403 PCSK9 (proprotein convertase subtilisinlkexin coronary heart disease AMG 145 type 9) (CHD) or high risk of CHD Evolocumab hvperlipidaernia hyperchole sterolaemia stroke atherosclerosis a cardiovascular disease or condition a condition associated with elevated LDL VLA 2 Inflammatory bowel SAR339658 Integrin a2Dl disease TL-4 Idiopathic pulmonary SAR 156597 IL-13 fibrosis lysophosphatidic acid receptor SAR100842 Systemic sclerosis LPA-1 fibrosis LPA-3 Androgen receptor Cancer, eg, prostate cancer, MDV3 100 breast cancer enzalutamide HERI Erlotinib EGFR Cancer. eg. NSCLC ERBITUXTM VECTIBTXnT VEGF receptor] ASP4I3O Cancer. eg. colorectäl VEGF rcceptor 2 tivozanib cancer, breast cancer VEGE receptor 3 JAK Inflammator disease. eg, ASPO15K JAKI rheumatoid arthritis Baricitinib JAK2 Diabetic nephropathy CD4O Prevention of organ ASP 1240 transplant rejection GnRI-I Endometriosis ASP17O7 Cancer. eg, prostate cancer degarelix PDE9 Lower unirar tract ASP49O 1 symptoms associated with benign prostatic hvperplasia TNF alpha Inflammator disease. eg, Certolizumab pegol rheumatoid arthritis, psoriasis. chrohn's disease,

IBD

Programmed cell death protein I Cancer Nivolumab Chronic myelocytic MK-3475 leukemia; Hepatitis C virus infection; Hepatocellular carcinoma; Hodgkins disease; Melanoma; Multiple myeloma; Non- Hodgkin lymphoma; Non-small-cell lung cancer; Renal cell carcinoma; Solid tumor; Stage JV melanoma Hepatocvte growth factor Cancer onartuzumab MET Glioblastoma; Hepatocellular carcinoma; Metastatic colorectal cancer; Metastatic non small cell lung cancer; Metastatic stomach cancer; Non-small-cell lung cancer Angiopoietin ligand-1 Breast tumor; Cancer; trebananib Angiopoietin ligand-2 Colorectal tumor; Fallopian Tek tyrosine kinase receptor tube cancer; Gastrointestinal tumor; Glioblastoma; Hepatocellular carcinoma; Mctastatic esophageal cancer; Metastatic gastrointestinal cancer: Metastatic non small cell ung cancer; Metastatic ovary cancer; Metastatic renal cancer: Ovary tumor; Peritoneal tumor; Transitional cell carcinoma CD37 modulator elotuzumab Cancer Lymphocyte ifinction antigen-3 receptor -Multiple myeloma SLAM family member 7 IL-2 dacliziLmab MuFtiple sderosis TL-2 receptor alpha EGFR Cancer necitumumab Metastatic non small cell ung cancer; Solid tumor IL-5 Asthma; Eosinophilic reslizumab esophagitis B-lymphocyte cell adhesion molecule Jnotuzumab CD22 Cancer. cg Acute lymphoblastic leukemia; inotuzumab ozogamicin Follicle center lymphoma; Non-Hodgkin lymphoma; epratuzumab Systemic lupus e rvth em ato sus. m oxetum omab hairy cell leukaemia moxetumomab pasudotox ILl beta Acne vulgaris; gevokizumab Atherosclerosis; Behcets disease; Cardiovascular disease; Dermatomyositis; Insulin dependent diabetes; MlLltiple myeloma; Osteoarthntis; Paraproteinemia; Polymyositis; Pyodenna gangrenosum: Scieritis; Uveitis CD2O Cancer Ocrelizumab Multiple sclerosis ofatumumab follicular lymphoma (eg, refractory or relapsed) diffuse large B cell lymphoma (eg, relapsed) chronic ymphocvtic lcukacmia (eg. first line therapy or relapsed) JL-23 Crohns disease; tildrakizumab Inflanimatorv disease; Psoriasis BAFF Autoimmune disease Belirnumab Neutrokine alpha systemic lupus BenlystaiM ervthematosus Tabalumab Multiple myeloma vasculitis TL5 Asthma mepolizumab TL6 Inflammatory disease sirukumab rheumatoid arthritis Lp-PLA2 Atherosclerosis darapladib diabetic macular oedema CCR9 chemokine receptor Inflammatory disease Vercirnon rheumatoid arthritis Crohn's disease DOPA decarboxylase Parkinson's Disease Patrorne Hcr2 Cancer. eg, gastric cancer, TyvcrbTM EGFR breast cancer, head and TykerbThl neck squamous cell cancer, lapatinib ADP receptor Cardiovascdar disease or Brilinta condition Brilique Thrombosis. eg, arterial thrombosis VEGFR Cancer. eg. Caprelsa EGFR medullarv thyroid cancer LABA PTOO3 GFF Respiratory disease or

LAMA

condition.eg, COPD Factor Xa thromboembolism apixaban Oxelumab Asthma 0X40 ligand Graft-versus-host disease Tremelimumab Cancer. eg. melanoma CTLA-4 Ticilimumab CD152 ipilimumab Autoimniune disease MPDL328OA Cancer. eg. solid tumour, MED14736 kidney cancer, lung cancer, melanoma. N SCLC. PD-Li

multiple myeloma Autoimmune disease Nivolumab Cancer. eg. solid tumour, AMP-5 14 kidney cancer, lung cancer, AMP-224 melanoma. NSCLC. PD-i

multiple mydoma Autoimmunc disease

LIGHT

TNFSFI4 CD4O ligand JAK Inflammatoiy disease. cg, tofaeitinib rheumatoid arthritis, psoriasis, chrohn's disease, IBD. ulcerative colitis, Psoriatic Arthritis Nerve Growth Factor Pain tanezumab Osteoarthntis pain Her I receptor dacomitinib Cancer. cg. Non-Small Cell Her2 receptor Lung Cancer Her4 reccptor c-MET Cancer. cg. Non-Small Cell crizotinib ALK Lung Cancer Programmed cell death 1 receptor Cancer. eg, renal cell Nivolumab carcinoma SLAMF7 Cancer. eg, muFtiple Elotuzuniab CD3]9 rnvelonia CD3O Cancer. eg, Hodgkin Brentuximab hrmphoma. systemic anaplastic large cell Brentuximab vedotin lvmphoma. T-cell vnipboma GPR4O Diabetes. eg. diabetes Fasiglif'am DPP-4 mellitus trelagliptin VEFGR-l receptor Motesanib VEFGR-2 receptor Cancer. eg, non-squamous VEFGR-3 receptor Motesanib diphosphate non-small cell lung cancer

PDGFR cKit

amyloid 13 Alzheimers disease Solanezurnab TNF alpha Inflammatory disease, eg, SIMPONfM rheumatoid arthritis, HUMIRA'-" psoriasis. chrohn's disease. REMICADEThI IBD, ulcerative colitis, ENBRELTM Psoriatic Arthritis Adalimurnab Table 5: PCSK9 SEQUENCES FGWvI/ALLELE SE411D £O.

J*MfPO 40 D SEQUEN CES = sjgnS sequence 1fl courier=ropept*de3132 -catdy?c donw 153-449 UPPERCASEC-term ra dova45c-692 Uidernd =rethes thangedfroma &e a - tAhe ces{a resdt rmbers o a PraEornwrr sgr seauece MGTVSSRSWV PLDLLLLLLLLLGPAGA9AEL EDEYEELVIALRZEED3LAEA?EEGTTATFH&CAYDP r ILVFi{GLLGFLVS3JFLALFLPF 7211EEE55 Jr spwnetapryrac qpggs 1sqsd"eeg adfevpeedgthqakcdshg tb agvsgdagvakg5rn sfrv ncqgcgt5gt ge rsa qpgp w pggvn aacra agw vtaagnfr&ac fapge drfsa 425 43 474 dvLB'; ed kpnhcaspv4 3AGWQFRvt1⁄4S-tSGPTRMA ALARCAPDEELL5C5SESRSGVR3EM LTGrss? a EDLGT S3 &20 H PPvLPPG 4E0<VKEIB PAPQVJ\AC(EGVflLTGCSQGtSHVLGAY 5:2 4\r a 2 wRLTi7xETHLsQszRTABpLc2oasYLTyILavrHGLLpcFL'> rMsG:(LLrLALELPIi VDiIEZtS7F±Qsuw'& ppyrscqppiggs ev$ &t s qdreeg' mtdfenvpeedg1rftrqaAccSi; ft' agngdavokgnsh ncqgkgtgjp qpvgj paggysrv 4 spsBvtvgatnaqcr4pv' g*gtnrevd pgEtg4csdcctcfsqcgtqaav3)R3 rn&aeptaefrq'1r'fse 42S 443 47' deapedq-ftpr1vaapasthC GVFcR S4HGPTSMATn CAPDEELLECSSFSF5GKRFGER\ _________________ _______________ EACEG'hCRAH rnFGGEG\ jCCLLPGANcS1⁄4n-rAaPAEAsMGTRVHCHQQGH vLTGrs5HEJEDLGT 615 623 AVDNTCVWSRDV.SITSSTSEEAVTAVACCRSgHL&2ASQELQ a MathreJorrn 3 asspEcjtgar;doptgt gs,srJcsc sqsgts;aam'agaarnn saepe iaErqr tsak 425 443 74 dVnEaWfpedqr4t9nppSthGAC-WQLFCRTVWSAHSGPTRMATMPRCAPDEELLSCSSFSSGKRFH3ERM HgpPVLRPRGcwNQCvt3HREA. sHAsCCHAPGLEcxVKEHCpAPQEQvPi4CEEGWThTGcsALPCTSHVLSAY A WEC V V VSUGSTSEEA VTA. VAC C RS RHLAOASQE LQ f 4 Sgna Secence MGfl5SRSWWPLPLLLLULLLGP4GM4QEDEDGDYE.ELVLXERSEEDGL7iAPEEGTTATFPPCAF.DP wRLPGTrnTTiuQQR:.RLQAQaAPY03nKiLiivGLLGnmcGDLLaALz.L FE t 425 445 474

EAQGGKLVCRAH

61.9 620 HKV1R:GQPNQCVCHEASj4SCCR&PCLECXVKHGP4PQEQVfVAC{EGWTLTGCS,4LPGTSHVLCM WDt(1C'VWLSDVST1GSTSEEAVTAVACCRSRHthOAS3.ELQ f Pro-Form QEDEDGEEL'ALRSEDGL7flPEHGTTATflTRCPZDP 5 VD7I _______________________ _____________________ 425 445 d EQ5GKLVCRAWAF6GE<3VffiARCCLLPQANCSVHThPPAEASM6TRVHCHQGGH.LTGCS5HWEVEDL6T 63 62-3 S 70 A.VDNflVVRSDVSUGSTSEEVTAVACC5RHL4QASQELQ t p, j Mature form spas3pev-tvgtr aqdopvt tgtrfgrcgf pged gossdcs cfvsosgt aaagamrnsaeQe%aefrqr tsak 425 443 4)4 d EAQGGKLVCMHAFGGEGVYARCCLLQANCSVH1149PAEASMGTRVHCHQQGHVLTGCSS*HWEVEC'LGT 613 020 AVDTCVVRSDVSUGTSEEAITAVACFSRHthOAScELQ a 46 -u-i c Pro-Form wth a Seoier MCT/SSPPSWNF'PLiLLL&LG4GrnAQEDEnt1Z2LVLALPEEE3nAE1ah;?ThTZI1PCAKDP t

S

423 443 474 dvLneawfpedqMtpn;aappsthGAGWSCRTVWSAHSGPTMATM4RCAPDEELLSCSSFSRSGKRRGERM EAQGGKLVCRAFffAFGCEGVø3CCUYQANCWHThPPAEASMGTRVHCHQQ6HVLT6C:SSHWEVEDLGT 613 620 HKPPVLRPGQPNQCVGHRE4SSCCHPGLECKVKEHGPAPOfDVVJACEEGWTLTGCSWGTSHVLGAY c Pro-Form QEDE.DGDYEELLRSEDGLEAPHGTTA2?3TRCThKDP _______________________ ______________________ 4gvvcgrdagvakgacmrc.r

S

425 243 474 SiS HKPPVRRGcncCVGHRE4swi&5CCHAPGLEnwEHGpPQEa\rrvACEEGWTLTGCSMpGTsHVLGAY mDTC\r/RsRDVsiTGsTsEGAsiTAVNCCRsRH::LAQA5QELQ c q Ms.ttre orni 9 {h*agvsgfJaglakgan s vhcqgqtshg qp4pc'v gg2 naacra gvQethdg'fridaz ç 443 474 EAciGGJcRAHNAFGGECVThJMCCLLPGANCSVFniPPAEAsMGTRVH:HQaGHVLTGcSSHWEvEDuL7T KPPVLFWRGONQCVGHREkSft1A5CCHAPGLEOMEHGPAPQEQVrVACEEGWTLTGCSMPGTSHVLGAY AVDTcVVRSWVSflGSTSEGVTAV?dCCRSRH'LAcSQELO p Pro-Form with 53 Sgna Sequence MG t 425 442 474 d EAQGGKiVCR&NAFGGEGVYeARCCLLPNC:WFTAPPAEASMGTRVHCHQQGHVLT&CSS.HWEVED1GT c23 A.VDTCVVRSflDVSflGSTSEA'dTAVAKCRSRHLAQASüELQ p Pro-Form EDEDGELVR3DGLEAPEFA3TTATFHRC1⁄2XDP LHVFEGLIPGFLVF»=4SGDLLELAL t sa.ptarqrMtsak: 425 443 474 dvnea:wpedqrAtpaappsthGAGWQLFCRTVWSAH&GPThMATAyARCAPDEELLSCSSfSRSGKRa5EW4 6S520 HKPPVLRPR6QPNQCV@HREASH?3CCHJQ6LECKVKEH6PAPQEQVNACEEQW11T6CSQP6T51VLGAY AVDNTC.VVRSDVS1TGSTSEGA\dTAVACCRSR-&4QASQELQ.

MaUme torm 12 t

S

423 443 474 LTRH HOQC-h1'GCSSN NEVEDLGT HKPPVFPRG'NQCVGHRFt&HASCCHWGLECKVKEHGPAPCjEQV1VCEEGWflTGCSMPGTSHVLGAV

AVDNTCVVRSRDVSUGSTSEGAVTAVACCRSRHASQELO

p Pro-Form wth 13 na Sequnr MGWSSRRSV4c'PLPLLLLLLLLEGPAGARAEDEDGO?.EELVLALRZEE.DGLVEAPEHGTTATFHRCaTOP

V thbg

4.25 474 d EAQGGKLvcMHNAFGGEGvYARccLLPa4NcsvHTAPPAEAsMGTRvHcHQQGHvLTccsHwEvEDLGT AvDNTcJvRsRcdsrrGsTsEEtvTAvMccaRHLAaAsQaq p Pro-Fønn QEDEDGDYEELVL1RSEEDGLVPZhGTTATFBR2KDP 14 RLPGT7VViFZETHLSQZERTaRLQAQpGY.LT.ILH'G*LGFLV.K14SGDLLELAMLPi t

S

225 443 474 dvneawfpedq.itpnkaapps.thGAGWQ1FCRWW5MSGPTRMATAV4RCADEEUSCSSFSRSGKRRGERM EAQGGKLVCA3:.NAFGGEGVYAhRCCLLPWNCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSFW4EVEDLGT 13 522

H

vDiTcvvRsRDv5nGsisEEwFA:vAccsHLAQAsQaQ m ProFc.rmwth 15 Egra Seqe ce Gp'SqSWI'pLLLL LLGPAGAPAQE:ED'a: ZEL rSDE B1PGT1TVThrZgTELSQZERTAERLQAQAPRGYLTLcILFVFGLLPGFLVM5GDLLELALK LP1 tr agv' cg dgaksnrm ncL4gggt sgig*rr qhecDpM paggysnAr aceraragwKtaag3frdda&j

S

425 443 474 dvneawfpedqrc'ftpratppthGAGWQLFCRTVWSAHSGPTRMATAARCAPDEELLSCSSFSRSGKR!ERM EQGGLVGMNAFGGEGVYNARCCLLPWNCSVHTAPPAE4SMGTRVHCHQQGHVL1GCSSHWEVEDLGT 13 522 AvDNTcvvRsRDvsnGsTsEEAvTA:vAccsRHLac{AsQaQ m Pco-FomTt QEDE.DGDYEELAiRSEEGLAPEgGTAT?RCMDP 1 VDYIEE.DSV.FAQpwnEflprryr3deyqppdggSvefldtSiqid1. 1eEgrwtdfe2vpeedgtrthrq3SkcdShg t

-

425 443 EAQGGKLVCfl.&-NAFGGEGVYARCCLLPQANCWHTAPPAE&SMGThVHCHQQGHVLTGCSSHWEV! 21-3 522 HPPVL9PGGFt4QCVQHEREASft1ASCCHAPGLECK1(EHGPMQEQvTV,4CE {GWT1TGCSALP5T5HVLGAY S7C

AVDTCVVRSRDVUGSTSEEVFAVMCCRSRHLLASQELQ

rn Mature farm 17 t 425 443 474 EAQEGKLVCRAHNAFGGEGVYRCCLLPQANCSVH.ThPP4EA;SMGTRVHCHQQGHVLTGCSSHWEVEDT AVDNTCVVRSRDVSUSSTSEEAVLAVMCCRSRF1LAQASOELQ Pro-Form wth U S g"& Squene MGfl/SSRPSWWPL0LLLLLLLlGP&3APAQEflEDGDYZZLflAixEEDflZAPP2GTTaT iflC7YLP w -V (31 tNagnsgrdagv3rgasrnr)rvb cagkg*ngt g 1 ksqvr gpM a agg,6raacq1a'agvvvtaagnrcidady 425 445 474 dae3:wfpedrtpaatppsthGAGWFCTWlSAHSGPTRMATAVMCAPDEELLSCSSFSRSGKR, GERM EAQGGKLVCRAHr4.AFGGEGVYARCCLLPQANCSVHTAPPAEAsMGThvHCHQ. QGHVLTGCSS*HWEVEDLGT 6232O HKPPVLRPRGOPNQCVGHREASHASCCPGLECKVKEHGPAPQVfVACEEGWTLTGCS.ALPGTSHV'LGA AVDtflCVVRSRDS/SUGStSEEAViAVACCRSRFilAQASOELQ B Pro-Farm QEDEDGDYEEL\SEEDGLPEi2'TATFaKCIXDP 19 U agv sgrdag 3gRmhvrLcgkgvsgt efk.7qvop gpkv a ggys?vnaaccrarg;tagi #25 443 474

_________________ ________________

EAQGGKLVCRAHNAFGGEGVThJARCaLPQANCSVHThPPAEAEMGTRVHDIQQGHVLTGCSSHWEVEDLGT S9 AVDNTCVVRSRDVSU&ST5EEVA.CCFGRAQ4SQELQ Mature form 20 thiagQvsgrd sgv8grsmrsrvfr cogkgtttsgthg etrksq t?qp'ph% waggfsrt? r8crorragsnKragrf ddR ly 425 443 474 dv sea EAQGGKLVCRAHNAEGGEGVMMCCLLPQANCWHTWPAEA5MGTFWHCHQQGHVLTGCSSHWEVEDUEJ a Pro-F*omiwth 21 Stga Sequerica WRLPGTiLFlETHLSQ3EPTAPflQAQGtLTRILHHC-LPGFLVfl4SGDtiEL7'flLPi (0 thgwsgrdagvgasms:vrogkgt sgthg C k v øggysEv passpevgatnaqd,,4 tgg lfgrvd th.gascdcstcfvsqsaaa'ag eaqinLae&t ae* or hfsak 4:5 443 474 DL;T VDNTCvVRSRDVSUG5TSEEAwAVAcCRsRHLczA5QELQ h Pro-Form QEDEnsDn.sLvLLRaEEDeLAKnEorTpTFnRorxDp 27 FJ' PGTWVVLETliLSQSERTARELQAFPGYLTKILhVFiiLLPGFLVYM3GDLLELAL thagwsgriagaKgan;' S vh'ccgkgtvsgthg e ksvq j*v maggvsfv sacq a' agvivtaagrft ____________________ __________________ s2asa7evrga aqdq'gig fgr: ; mmsae2etaefror 425 443 474 EAG GLVCRAJHNAFGGEGVYAJAR.OCLLPQANCSWTAPAE4SMCTRVHCF4OQGHVLTGCSSHWEVEDLCT fl9E2 5-74 AvDNTcvvR3-D6nGsTsEEA:vTAvAccRsRHLi1&sQELQ Msture fctrm 23 t 425 443 474 EAQGGLVCMNAFGGEGVYAJAROCLLPQC9J-fTAPPAESMGThVHCF1GQGHVUGCSSHWEVEDLGT HxppvLpRGQDcvGHREAsw1AscCHGLECKvKEHGpAppEcAivAcEB3WrLTc5csAP-GtsHvLGAy 574.

AVDNTCVVRSDVSTFGSTSEEAVTAVCCRSHLAQASQELQ Cl

C --

Cf Pro-FCrm wth 24 Egna Ssqueire T&TVSS5W'LPLtLLLLLLLGP9GAqAQEDELGL!EELJLALL3EEGLEL5ZGTflTF-'ROA5P 0 t 425 443 471 dvLn-eawfpedqrvtpthaappsthGA-GWQLFCWWSAHSGPWMATAVARCAPDEELLSCSSFSRSGKRRGER M EAQ-6GLVCFMN.AFGGEGVt4AFtCCLLPQANCSVHTAPPAEASMGThVHCHQQGHVLTGCS5HWEVEDLGT 4VDTCWRSDVSTiGS-TS-FE4VTAVACCRSRHLAOASOELQ Pro-Form QEDEDYEZLLaLLZZE.DGLAEAPEGTTAT.fliRcAKD2

____________________ __________________

426 443 474 EAQGGK LVCRkHFGGEGVYAMRCCLLPQANCWHTAPPAEASWGTRVHCHQQGHVLTGCSHWEVEDLT AVDNTOSVFSRDVSTICST.SEEAVTAVAKCRSRHLAQASQELQ q Prc4ormwth 25 sgna Sequenc6 MGPiSSRfSWWPLPLLLLLLLLLGP4GMAoEnEvc4n?EELv.Lr434zEznGiyEAPEi!*snATFHRc$nP 425 443 474 dvne.awthedqrtpntappstha&GWFCRTVWSA$-fSGPTF3.M. ArAARcAPDEELLSCSSF5RSGkRGERM EAQGGKLVCAHNAF3GEGVYAARCCLLPQANCSVHTAPPAEASMTRVHCHQQGHVLTGCSSHWEVEDLGT C) -s HKPPVLRPRGQPNQCvGHREAsH5*cCHAPGLECKVKEHGPAPcQVTvACEEGWTLTGCS.4LPGTSHVLGAY q Pw-Form QE.DEDGDrTEEL'LFZE.DGLVEAPEE3TTATfl9ROtiKflP 2?

WLPB t

S

429 443 474 [AQGGKLVCRA1NAFSGEGVVAMCCLLPQANCSVHTAPPAELSMGTRVHCHQQGHViTGCS5HWVEDLGT HKppVLRPRGQPNQCVGHRMSHASCCHAP*GLEflWXEHGPAPECVP1ACEEGWTLTGCSALpCTSH' _________________ ________________ AVLMgK.VVRSRDVSfFGST:SEGAVTAVACCRSRHL4Q&ScELQ N LW ftOTDE SEQUENCES ut-ucect4ese enceerüdmsgrSsecuencerit: ePdes1-rrjj;Ier r cntde ser UPflCE enwrnng p pep* e cdq1-455 ca;e= nudeotdeseq terienccdngcatetc3oma riuceatdes457-134& UPr[RC&SEr rc cVde sequnc2encothnC-ter1 ama+ruz tdes 1347-fl76 ATGGGCACCIiTCAGCTCC4GGCGGTCCTGGFGGCCGCTGCCACTGCTGCTCCTGCTGL GCTGCTCCTGGGTC 2S Cr CCGCGGCCGCCCGTG CS cAGGAC ADGAG GAD CS TAGAGGAG cit ST GCTASCTTGCSTTCDG I zCctoc AG SAGGACGGCCTGGCCGAGCMCCGASCACG'AACCADAZCCACCTDCCACCGCTGCGCCAäGGAT CC3TG3GTTJCCTGGcACr TACGTIGGTGCTGGGGAccCACCTorcr4CTcA3AGcs cADTGcccGrcGzCTGcAGcc?L&rTGcCrcccGGATAtTcAccAAGATCr:GcATGTcTTc

AT SGCCTTCTTCCTGSCTTCCTGGTGAASATGAGTGGCGACCTGCTGGAGCTGGCCTTGAAGTTGDCC

cGTreacTAcaTceaG2GacTrcDnGrcn?GcccaGagcatcccgtgpaaccLggcattaccctcc& cggtaccggcggatgatacag:ccccga:ggaggc&gtdggtggagg gtat t:ct&gacaccagcMac&g3gtgE4c:accggaa atcgaggcaggLcatL4ccgaLtcgagL atgtgcccgaggag&gggaatgcrdcagcagccgcadgtgtgacagtcat ggcacccctggcaggtgglcgcggccgggatgccggcgtggccaagg gccagc3tgcgcagc:tgcgcg'grt aactgccaggg aagg2cggttageggcaectcataggz tggagtttatteggaaaagtcagctg ccagcrtgtggggccactggtggtgctgrtgcecrt ggcggg ggtacagccgc t cgrgcrtgccsjcgcdggcgagggcgggt gtgtggt accgctgcggcecttccgjgar gatg ctgcctcta rtrcccagcctcgctcctaggtcatccagttgggccacca atgrctaagB ccagccggtgaccctgggga rtLtggg gacca»=ctttggccgdgtggactct1gccccagggagg; catctJgtgcctc2gcgactgcgcarrtgctttgtgtcragagtgg gacatcacaggctgctgcccacgtgtgc3ttgcajrcatgtgctgtct ccgagccgg3jctcerctggrcgagttgaggcagagactg N2SSMTta X31 c2ccc2t3GGGCAGGTTGGCAGCGTflTGCAGGNLT3flSGGTCAGCPCMLTCGGGGCCTACACGGATGGCC 4'8 Ct,i'C hcA6CcAT6cccGcTGcGccccAGATGt.GGAGcTGcTGA3cTGcTCcAGmcrccAG6AcTGGa&4GcGG C&GGGCGAtC&CATGGAGCCCAAt3CEGGCAASaGuTCTGCCGGUCCCACMCGCTflTBGG@61 TGTCTACGCCAUGCOGGTGCGCCTGCThCCCCAGGCCMCTGC,GCt5TCCACACAGCTCCACCG 6CA3CAT66&6ACCCGTGTCCThCCACCMCA6&5CCACGTCJCAC&6CTGCA3CTCCCACTC ________________ ______________ GTGGAGGACCTrGCOCCCACMGCCGCCTG GCTGA3GCC4CGAGG CAGC tAACCACTGCGTG2 AGGGAGGCCAGCATCCACGCUCCTGCT&C.CATGCCCCAGGTCTGSAATGCAMGTCAAGSAGCATGGAA *ft*6CT ES4t333

I ______

T6GGAccTcc.cAcGTccTGGGGGccTAc&c.cGT. AAcMfAcGTGTGTAmcAGGAGcLG*GGAc2TCAGcA E711? *CTAcAcGcAe:ACcN3csAtcAcGccGTGAcAsccG1TGccATcTccTGccGa&sccGscAccT6GcGcAcG

CCTCCCAGGAGCTCOGTGAC

f ATGGGCACCGTCAGCIcCAGGcGGTCCTGGTGGCCGCTGCCACmCTGCTGCTGcTGaGCTGCTWTGGGTC R4SLCaVtc CIT CCGCG.GGCGCCCSTGCGCGGDG&GAcGGcGcG7.jGaG:TGGTGCThGccrTG*cGTTccG 53VeCCtcaPC CCGTGGAGGPTCCT GGaCCTZG*GGTGG2GCTGAGGAGGAGACDC%:CTCTCGCGTCAGaGCG ATGccTTcTTccTGGcTTccTG3TGAaT*Gj3*TGGcGAccTGcTGaC4cTGGccTTGaAGTTrsccc cAT3TcGAcTAcATcGaGGGArccTcrGTcnTeccckGagca.tcccgtggcctggagcggattaCccc{cca. (A)

JgcggflgggtacagccgcgtcctcaQgccgcdgccagcgcctggcgagggctggggtcgtgdggtcaccctgc cggcaathccgggac N42STwA'iT :C3CCC3tGGGGcGGUGGCAGCTGflTGCAGGACIGTATGGTCAGCACCICGGGGCCT4CACGGAIGGCC ACAGCCGJCGCCCSCTGC.GCCCCAGAT6GGACTGCTGAGCTGCfCCM3ITFCTCCAGGA&TSGAAEO3E CGGGGCGAGCGCATGGAGGCCCAAGGGGGCAft4CTGGTCTGCCGGGCCCADWGCTmGSGGGTGASGG TGTCTACGCCAITGCC4GGTGCTGCETGCTACCCCAGGCCMCTGCAGCGTCCACACAGCThCACC4GCTGkG GCCAGCATGGGGCCCGTGTCCgCTSCCACC&4CAGC,GCC4CGTCCTCAC&3GCTGCGC:ICCCACTGG

GTGGAGGACCUGGCACCCACAACCCGCCTGTGCTGAGGCCACGAGGTCAGCQTMCCAGTGCGTGGGC

QS9PC4GtCC& E62saAsto&33 TCCCGGCCCCICAGGAGCAGGTGACC:STGGCCTG:CGN3GAGGGCTGGACCCTGACTG&CTGCAGTGCCCTCC *cp3GGAccTcccAcGTccr66GoGccTAcoo3TAGAoPAcAc:6T6T6TAcTcA66AGcc65GAo3TcAGcA Ef7JGa43 tc.G *CTACAGG*CAGCACGaCQGMGAGGCCGTGACAGCCGUGCCATCTGCTGCCGGAGZtGGCACCTGGCGCAGG ________________ ______________ CcTCCCAG:GAGcTCC.AmGK _______ ATGGGCACCGTcAGC?cc4G. GCGGTCCTGC-TGGcCGCTGCCAcTGCTGCTGCTGCTGCrGCFGCTCCTGGGTC 3C R4.ELCGTt.CU

C

ATGGCCflCTTCCTG3CTTCCTGTGAP2ATGA3TGGOGACCTGCTGGGCTGGCC.TTGAAGTTGCCC CATGTCGACTACMtGAGakGGACTCCTCGTCTTTGCCCAGSgcatCccgtggaacctggagcggattacccctc ca a -ggcggggggtacaccgcgtcctcaacgccgcctgccagcgcctggcgaggg*ctggggtcgtgctggtcaccg ctgccgcaacttccgggac g 42SMTtA3T 44TCCCtCctC *c;cccatGGGGCAGG1TGGCAC-CTGI1TFGCAC, GACTGTATGGTCAC-CACACTCGGGGCCThCACGGATGGCC cG*GGGcGA&cGcATGGAGGco:AA&GGGQAAG*crc-GTcTGccGGGcccAcAAcGcTfTrGGGGGTG4GGG TGTCTAcGCCAHCCcA&GTGcTGccTGCTACCcCgJ3GCcMcTGCAGCGTCCACACAGCCCACCAGCqGAG GCCAGCATGGGGACCCGTGTC*CNT&CCACCAAcAGGGCCNGTCCTCAC4GGCTGCAGCTCCCALTGGGAG GTGGAGGACCUGGCACCCACMGCCGCCTGTGCTGAG000' CGAGcrcAGcCCAAccAGtGcGTGGer AGGGAGCCC&GCATCCACGCTCCTGCFGCC4TCCCCCtGGTCTGC<AATGCAMGTCAAGGAGCATG( QSPCA&M.CC& E62OGG?3tcGGG TCCCGGCCCCTC4GGAGCGSTGA.CC3TGGCCTGCGAGGAG&GCTGGAQITG1CTG&CTGC43TGC CTGGGACCTCCCACGTCCTGGGGGCCT.ACZ&CO5TAG.ACMCACGTGTGTAGTCAGG46CCGG&CZ3TCAGCA CTACAGGCAGCACCJGCGMGGGGCCGTGACACCCGTrGGATOTGCTGCCGGAGCCGGCACCTGGCGCAGG

CCTCCGA GGAGCTCCAGTGAC 31

R4SLC.izeCfl

C

AS 3CC to PGGGGc*GGc.cGGDcathGDccGAGcac*GAADcAcAGccAcc.nccAc*D;cTGo;c*caAGGAT

C

CACIG

A

C

cggtCCgggCggtgataCcagCccccCgaCggaggcagectggtgg2ggtgttdCctagacBccagcatcagag tgacCaCcgggEa g aagggcaggtta. gcggc3ccctc3taggccggagtttattcggaaaagCcagctggtccagcctgtggggcGctggtggt. gctgct.gcccct ggcgggtgggtaca. gccgcgtcctcaaQgccgcdgcegcgcctggcggggdggggtLggctggtcsccgLtgccggcsacttccggga c C) gcatcacaggctgctgcc*cacgggctgcattgc3gccatg3tgctgtctgccgagccggagcecaccdgccga gttgaggca3agactg S55AXto accacttctctgccagatgtcacaa1gaggcd: ggttccctgaggaccs*gcgggtadgacccccaBcctggtggccgc{gccccccag cacccatGGG6CA:3G1TGGCAGCTGTmGCA6GACTGTATG6TCGCACACTCGGGGCCT4CACGGAfl3GCC 474ATCte3TC ACAGCCG1CGCCCGC1GCGCCCCAGATGAGGAGCTGCTGAGCTGCT:CCAGTffCTCCAGGAGTGGG4iGCC,Q CGGGGCGAGCGCATGGX,GCCCAAGGGGGCAAGCTGEFCTGCCGGGCCCACAAcGCTTTTGGGGGTGAX3GG TGTCTAU3CCAUGDCAGGTGCICCC.TGCTACXCAGGCGACTGCAGC.GTCCACACAGCTCCACCAGC.TGAG GCCAGCATGG:GG4CCCGTGTCCACTGCCACCMCAGGGCCAC6TCCTCACAGGCFGCAGCTCCCACTGGG43 GTGGAGGACC1TGGCACCCACAGCCGCCTGTGCfGAGECCACGAGGTCAGCCCA/CCAC'TGCGTGGGCCAC ACGCAGGCCAGCATCC4CGCITCCTGCTGCCATCCCCCAGCTCTGGNTGCAMGTCAAGGAGCATCthC Q19PCA3 LoCCC E520t3 a43tc 3 TCCCGGCCCCTCAGGAGCAGGTGACCGTGGCCTGCGGGAGG.GCTGGACCCTGAC1GGCGCAGTGCI ________________ ______________ CTGG6ACCTCCC4CTCCTCG6&3CCTACGCCGTA<3JCMGACGTGTGTA.6TCAACC5(3GACGT( cT.AcAGGcAGcccAGcGM.GGGGcaTGAcA.GccGrrGccATcTGcTcccGGAGccGGcAccTGGcGcAGG CCTCCCA*GGAGCTCCAGTGAC p 4 TGGG cCGTcAGOTCcAGGCGGFcCTGGTGGcIGCTGcCACrGCTGCTGETGcTGCTGCTGCTCCTGGGTC 32 g46LCT tt CIT

C

AS3VGCCtzCIC

A

cAcTccCeccGccTGcAGGcccAG3cTGcccGccG3e3ATAccTcAccaAaTccTGoATGTcTTcc cggaccgggcggatgaathccagccccccgacggaggcagcctggtggaggtgtatdcctagacaccagcatac agagtgaccaecgggaa a ggcacccacctgg*c; ggggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgca*gcctgcgcgtgctca; ctgccaggg aagggcacggttagcggcaccctcataggcetggagtttattcggaaaagccagctggtccagcctflggggca ctggtggtgctgctgccect.

ggcgggtgggtaca. gccgcgtcctcacgccgcctgccagcgcctggcgagggctggggEcgtgctggtc3ccgctgccggcaacttcc gggac gaccaactttggccgctgtgtggacctcttgorcgggaggacat*c; tttgcctccagcgactgcagcacctgctttgtgtcacagtgg g r42MTtAGT cBcccatGGGs.c4GGU6GcAccTGmGcAGGAcTGTATGGTc,GcAcAcTcGGGsc:ct4cAcGGATGGcc 474VAIttc3TC ACAGCCGTCCCCC&flGCGCCCCAGATGAGGA:GCGCTGM-CTGCT:OC.&GTflCTCCAGG&GTGGGMGCGG CGG&GCGC&CATGGA&GCCLAAGGGGGCAAGCRG1CTGCCGGGCCCACMUC1TUfGGGGG1GAGCG TG:TCTACGCCAflGCG6TGCTCCTGCTACCCAGGCCMCTGCAGCGTCC4CACWCTCCACCA.SCTGG &CCAGCATGGGG&CCCTGTCCACTGCCACCAACAG&SCCACGTITCACAGGUEGCAGCTCCCALTGGGAG GTG3GGcc1TEEc4cccAcMGccGccTGTGcGAGGccAcGAcTEAGcccAAccAGTGcGmGGccAc A&GEAG&ZCACCATCCACGCf{CCTGCfGUAiCCCCA{3GTCTGGAATGCAM5TCAGGMiCAIGG&& Ugt9FCA3z.CCG E2C3AGt:GS TCCCGCCCCCCAGG&SCAGGTGACCGTGGCCIGCG AGE AGGGCTGGACCCTGACTGGCTGCAGTGCCC _______________ ______________ CTCAEGCAGCAccACcGflGAGGCCGTGA.cAGC:CGTrGCCATCTGCTGCCGGAGCCCG CACCTGGCGC

CCTCCCAG S AGCTCCAGTSAC

p3.. TGGGcAcCGTCAGcTCCAGGCGGTcCTGGTGGCCGCTGcc4CTGCTG'TG&GCTGUGCTGCTcCTGGGTC 33.

F4 L C3Tt. Cfl CCGCGGGCGCCCGTGCGa&GPJsac.GA3GAcao:GIfTacGAGGAGcTGGTGcnGccnGcGTTccG 45'JSCCta&C 7sGAGSMGGccTGGocaGcAcccGztGcAcGGAccAcAGccAccTTcc2ccGcTGcGccAAGGzT ATGGccTTcTTcc.TGGcTTccrSTGAASArGAGTGGcaCcTGcTGaGc.TGGccTTGJGI'Gccc ACAT GGATCflCBflC TTTGCagctcccgtggaacctgagcggatacccctDc* cggtaccgggcggagaataccgccccccgacgaggc8gcctggtgggtgatctcctagacacc&gcatacag. agtgaccaccggaa atcg2gggcagggtcatggtcaccga*cttcgag2Jtgt, gcccgagg2ggacggga*cccgcttccacgacaggccgcagtgtgscagt*cat agggcscggttgcggcaccctcataggcctggagtttdttcgga.Egccagctggt*ccagcctgi, gggccactggtggtgctgctgcccct gatgcctgcctctaetccccagcctcagctcccgaggtcateacagttggggccaccaa. tgcccagaccagceggtgaccctgggg3ctttggg gacatcacactgdgcccacpjggctgcattgcagccatg3tgctgtctgccga: gccggctcccctggccgagttgggcsgagadg 42S5MTIc5GT,A443TKCtaCC caccctSGGGCACS1TGGCAGCT6TfflGCASGACTGThTGGTC.&CCAC*CTCGCCSCCTACACGGATGGOC 4?4VA}ttpi3TC ACAGCCATC.&CCCGCTGCGCCCC/43ATGAGGACCTGCTGAGCTGCTC:CAGmCTCCAGGAGTGSGAAGCGG GccAGcATGGG&AoccGTGTcccTGccftca&gcAGS6ccAc.cTccTcAcAGGcTacAGcTcccAcTGGGAG GTGGAG*GACCITGGCACCCACMSCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCAC ASGGAGGCCACCATCC:ACSCITCCTSCTCCCATGCCCCAGSTCTSGAATGCAAAGTCAAGGAGCATSGAA Q3Pt.3taCC E2AGtzGS TCCCGGCCCCICAGG.&GCAGSTGACCGTGGCCFGCGAGG4SS*GCTSGACCTSALqGGCIGCAGTSCCCT*cc CTGGGACCTCCCACGTCCTSGGG&CCTAQCCST4GACMCAC*EJGTGT4GTCAGGAGCCGGGACGTCAGGA CTACAGGCAGCACC:AGaAAGAGSCCGTG4CM5CCGUGCCATCTGCTSCCG3AGCCGECACCTGGC&

CCTCCCAG GAGCTCCAGTGAC

e ______________ ?TG&GC4CCGTCAGCTCCAGGCGGTCCTGGT&GCCGCTGCCACTGCTGCTGCIGGTGCTGCTGCTCCTQG Cit 4S3V eCCtOc.TC

C

ATGGCCTTCTTCC.TGGCnCC.T1rTG.aGAGMGGCGACCTGCTGGaGcTGGcCTT.AGThrCCC a gcacccacdggcaggggggtcagcgctgggatgccggcgtggccaagggtgccagcatgçgcagcotgçgc, gtgct caactgccagg ggcgggtgggtacagccgtcctcaacgcgcctgccagcgcctggcgagggct, ggggtcgtgctggtcaccgdgccgcBa.cttccgggac atgcctgc.ctctactecccagcctcagctccgaggtcatcacagttggggcca: ccaatgcceaagaccagocggtgaccctggggacttggg ATt4GT.4flCCt.r&D caccc&:GGGGCAGCUGGCAGCTGTVflGCM3GACTGTATGGTCAGCACACTCGGGGCCTACACCGATCGCC 74 to ST QD ACAGCCGTCGCCCGCTGCGCCCCW3XFGAGGACTGCTSAGCTGCTCCAGrnCTCCN3G4GT&SGMGCGS CGGGGCGAGC:GCATGGACGCCCAUSCGGGCAAGCTGGTCTGCC&GGCCCACMCGCBTrGGGGGTGAGGG GCCAGCATGCGSACCC:GTGTCCACTBCCACCAACAGGGCCAC3TCCTCACACCCTGCAGCTCCC,CEGGGM3 GTGGAGGACCTGGCACCCACMGCCGCCTGTGCIGAG&CCACGAGGTCAGCCCAACCAGTGCGTGGGCCAC AGGGAG3CCAGCATCCCGCnCCTGCrGCCATGCCCCN3GTCT&SAATGCAMGTCMGGAGCATGatA Qi9PCLaCO3 EE23:3t33 TCCCGGCCccTCAGGAGCAC*GTCACCCTGcCCTCcCAGG&CGCTCG.4cCCTGAcTGCcTGCAGTGCCaCc cTGGGAcCTcCcAcETCCTGGGGsCcT4cGcCGTGAc&4ctcGrGTGTAGT*CsGAccCGG6ACGTcAGcA CTACAGGCAGCACCGCGM6AGGCC:GTGACAGCCGTTGCcP,TCTGCTC-CCSGAGCCGGCCCTGGC.GCGG CCTCCCAGGA.GCTCCAGTGAC h ATGGGCACC*GTCAGCCCAGGCGGTCCIGGTGGCCGCTGCCACTGCTGUGCTGCTGCTGtTECTCCTGG R4 61C&t ASS 3CC t GTC A1G CGGC OT:C CCG' CA000: GCACGGA.A CCAC CC AC OTT CCACCtCT:G0000Ak GAP

C

*.c,rTcc

A

cggt:accgggcggatgaBtaccagccccccgacggBggcagcctggt:ggaggtgt. 3tctcctg&caccagcat.3cgtgaccccgggaa ggca:cccacctggcaggggtggtcagcggccgggatgccggcggccaagggtgccagca {gcgcagcctgcgcgtgctcaadgccaaggg aQgggcacggttagcggcacccr.cetaggccggagttsttcggaaeagccagctgt. ccegcctgtgggcc&tggtggtgcgctgcccct ggcggtgggt;cagecgcgtcctc. cgccgcctgcczgcgcctggcgaggetggggtcgtgctggtcaccgctgcegcaacttecgggac gatgcctgcctctactcccc&gcctcagctcc*cgaggc*stcacattggggccacca*atgcccaogaccacc ggtgsccctggggsctttggg gacat.ca*cggcEgctgcccacgtggctggcattgcu3gc*c2tgatgctgctgcc. gagccggagctc3c*cctggccgagttgaggcagagac1g 4443T3CCrACC c3CCCatGGGGCAGGTFGGCAGCTGT1T [TGCAGGACTGTATGGTCAGCACAGTCGGGC'CCTACACGGATGGCC I474VATtSZ?GTC AcAGCCATC*GGCCGCTGCGCCCGAGMGAGGAGcTGCTGPGCTGcTCcAGmCTccAGGAGTGGGMGCGG CG.GG.GC:GAGCGCATGGAGGC: CCAAGGGGGCAAGCIGGTCTGCCGGiSCCCACMCGCTVflGGGGGTGM3GG TCT*CTAGCC*cnGcCAGGTGcTc<CTGCTACCccwGccMcTGcAGCGTcctCACAGCrCcA*CcAGcTGAG CCCAGCATGCG6ACOCGTCTCCACTCCCACCMCAC.GGCCACI3TCcTCAcAGGCTGCAGC.TOCCACmGGAG AGGGAGGCCA&TCCACGCflCCTGaGCCATGCCCCGGTCTGGAtTAA4GTCMGGA&CATGtA Q4tFC,3t>CCG E62OGe43t., TCCCGGCCC.Tt&GCAGGTGACCGTC:GCCTGCG$CGN300CTG&CCCTGACICGCTGCAGTGCCCTCC CTGGi3ACCTCCCACGTCCTGGGG&tiACGCCGT4GA.CAAC4CGTGTGTAGTCAGGAGCC.G: GGACGTCAGC4 t>n.

CTh:CA:GGCAGCAIAGCGAAGAGGDCGTGCA&CCGTFGCTATCfGCT&CCGGGCCGGCCCT&GCGCAGG

CCTCCCAGGAGCTCCAGTGAC

ATGGGCACCGTCAGCTCC4GGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCGCTGCGCTGCTCCTGGGIC 36 A53 V GCC tGEC:

A

CCGTGGAGGTGCCTGGCACCTACGTG2IGGTGZTGAAGGAGGAGACCCACCTCTC.GCAGTCAGAGCG GGCGADCTGCTGaGC'GGCTTGAGTTGCDC *cATGTcGAcTcacGAG. GAGGAcaccTcT*sTcTTGcccaGagcicccgtgga2cctggagcgattacccctcc2 cggtaccgggcggatgaataccsgccccccgacggaggccctgglggaggtgtatctcctagacaccagcatac agagtgaccaccgggaa ggcacccctggcaggggtggtcagcggccgggtgcggcgtggcca3gggtgccagGtgjgc2gcdgcgogtgct caactgc*caaggg ggcgggt:gggtacagcegcgccteaacgccgcctgccagcgcggcgagggct: ggggtcgtgctggtccegctgccggcacftccgggac g gaccsactUggccgtgtggatcittgccccaggggagacatcatt, ggtgcdccagcgactgcagcacctgcUtgtgtcscgagtgg N42E Ml t A3l M431 (iCC tc ACC *atcCacttctctgcc2a2gatgtcatC4gBggcctggttccctg3ggaCcagcgggt2ctgaccccCaaCCtg gtggccgççCtgcccCcCBg :CacCC&GGGCAGGUGGCAGCTGfl1T&ftGG.ACTGTATGGTCAGCACACTCGG&GCCtACACGGATG43C.C ACAGCCGTCGCCCGCTGCGCCCC.AGATCAGGAGCTGCTGAGCTGCTCCAGTUCTCCAGGAGTGG&AAGCGS

C -

mTcmcGccAnGccAGffrGcrGccTecTAcccc1AGGccMcrscAecGTccAcAcAGaccAccAGcTaAG GTGGAGGACCITGGCkICACMGCCGCCTGTGCTGN3GCcACGAGCTCAGCCCAACCACTGCGTGGGCCAC AGGGA&iCCAGCATCCACGCHCCIGCIC:CCATSCLLCAGGTCflGAATGCAAAGTCAAGAGCAJGGAA Ql9PCAtCC3 E2k(i43& TC:CCGGCCCCTCACGAGCAGGTGAC:CGTGCCCTGCGNGA36GCTGGACcCT6ACTGGCTGCAGTGCC:cTCC CfGGGACCTCCCACGTCCT&GGGGCCTACGCCGJWACMCACGTGTGTAGTCA&GGCCGGGAWTCAGCA CTAC.4GGCAGCACCAGCGMGAGCtCCGTGACAGCCGUGCCATCTGCTGCCGGAGCCGGCACCTGCCGC4GG

CCTCCOAGGAGCTCCAGTGAC

ATGGGCACCGTCAGCTCcAGGCGGTCCTrSGTGGCGScrGCCAcFGUGCTGcTGCTGCTGCTGCTCCTGGGrC 37 R4LCi3ltGCU CCGCGGGCGCCCGTGCGcGGACCGJGGJtCGGcGAcTACGLGGAGcTGGTGaPGCCTTGaGTTCcG 437VGCC tzGTC

A

CAT Tc:CaCTAcPTC*aSGk3GACTCCT*c:*GTcnTGcccksagcatccqtggascctggagcflattscccckcs cggtaccgggcga:gaataccagccccccpcggaggecctggtggaggtgatctcctgcaccB.gcaca. gagtgaccccgggaa g aagggcacgtagcggcaccctcEtaggcctggagtttattcggaaagccagctggtccgcctgtggggccactg gtggtgctgctgcccct gcgggtgggtacagccgcgtcctcaacgccgctgccagcgc*ctgcgagggctggggtcgtgctggtcaccgct gccggcaacttccgggac gatgcdgcctctact*ccccagcctcagctcccg2gtcatcacagtcgggccaccatgcccaapacc3gccggt gccctggggadttggg gacc: actttggccgctgtgggacctdttgccccaggaggacatcattggtgccXccagcgactgcagcacctgctttg tgtcacagagtgg gac2tcacaggctgctgcccacgtggctggcattgcagccgatgccgtctgccggccggagcicaccctggccg agttgaggc2gagctg 42ATt.43T M1reccQ3c cacoatGGG I3CA*GGflGGCAGCTGTI1TGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC cAGcCArcGCD:GCTGcGcCDcAG4Ta4GGAGCTGcTGAGCTGcTCCAGmcrCCAGGAGTGGG.MGcGG CGGGGCGAGCGCATGGAGGCCCGSGGGCMGCrGGTCTGCCGGSCCCACMCGCTm&SGGGTGAGGG TC?TCTACCCCATfGCGA:GGTGCTGCCTGCTA. OCCCAGCCCMCTCCACCGTCCACACAGCTCCACCAGCTCAG GCCAGCATGGGGAcCcGTGTCCACTGCCACCAAcAGGGCCCGTCCTCACAGGCTGCA3cTCCcAcTGGGAG -GTGSA&iaccusGcAcccAcnsccGccTGrsacAsGccACGAGGTCAGccCMCCAGTGcCCGECCAC A.GGGA.GGCOJ3CATCC,sCGCUCCTGCTGCCATGCCCCAGGTCT3GA4TGC4M3TCAAGGAGCATGGM QPCACtcCtC 2XtrCiGZ TCCCGGCCCCTCA:GGAGCAGGTGACCGTGGCCTGcGAGGAGGGCTGGACCCTSACTGGCTGCAGTGCCGCC CTGGGA.CCTCCCACGITCTGGGCGCCTACGCCGTAGACAACACCTGTGTA.GTCAGGAGcCGGGACGTCAGCA CTACAG*GCAGCACCAGCGMGGGGCCGTGCAGCCGTtGCCATCTGCTGCC*GGAGCCGGCACCTGGCGCAGG

________________ ______________ CCTCCCACGAGCTCCAGTGAC _______

Table 6: Human VH3-23 Variant Alleles VH3-23 Cumulative allele haplotype frequency ___________ SNPs ___________ a (=VH323*04) 00983 rs56069819 ___________ ___________ ___________ d 0.0087 rs56069819 rs61750837 rs61752504 ____________ C 0.0046 rs56069819 rs1064090 rs055799 ___________ ________________ 0.0009 rs56069819 rs1055799 ___________ ____________ u 0.0005 rs56069819 rs1064091 ___________ ___________ s 0,0005 rs56069819 rs1064091 rs61752504 rs61750837 r 0.0005 rs56069819 rs1064090 ___________ ___________ TOTAL: 0.114 Table 7: Exemplary anti-PCSK9 antibodies and/or antibody fragments SEQ ID NOs comprising an anti-PCSK9 Patent or patent Pblication monoclonal antibody or fragment thereof Light chain complementary determining regions US2O 120020975 Al (CDRL) SEQ ID NO: 5.7,9. 10. 12. 13. 14. IS, 16, 17, 18. 19, 20, 21, 22, 23. 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 270, 271, 272, 273, 275. 277, 286, 287, 288, 297, 299, I, 405, 407, 409, 411. 413, 4 IS. 417, 42 I. 425, 429, 433, 437, 441, 445. 449, 453. 457, 461, 465, 469, 473, 477, 481, 485; Heavy chain complementary determining regions (CDRH) SEQ ID NO: 47. 48, 49. 50. 51, 52, 53, 54. 55. 56, 57. 58, 60, 61, 62, 64, 65, 67, 69, 71, 72, 74. 76, 77. 78, 79, 80, 81. 83, 85, 87, 89, 91, 278, 289. 290, 291. 292. 298. 300. 302, 401. 404.

406,408, 410, 412, 414,416, 419,423,427, 431, 435, 439. 443. 447. 451, 455. 459. 463, 467, 471, 475, 479. 483; SEQ ID NOs comprising an anti-PCSK9 Patent or patent Pblication monoclonal antibody or fragment thereof CDRL SEQ ID NO: 5. 7, 9, 10. 12, 13, 15, 16, LJS20120027765A1 17, 18, 19,20, 21, 22, 23,24, 26, 28. 30, 31, 32, 33, 35, 36, 37, 38, 39, 40,42, 44, 46, 405, 407, 409, 411, 413, 415. 417, 465; CDR}I SEQ ID NO: 47. 48, 49. 50. 51, 52, 53, 54. 55. 56. 57. 58, 60, 62, 64, 65. 67, 69, 71, 72, 74. 76. 77. 78. 79, 80, 8!, 83. 85. 87, 89, 91, 404, 406,408,410. 412,414,416,463; CDRL SEQ ID NO: 5.7,9, 10, 12, 13. IS, 16. US8 168762B2 17, 18, 19,20, 21, 22, 23,24, 26, 28. 30, 31, 32, 33. 35. 36, 37. 38, 39, 40, 42, 44. 46, 405, 407, 409,4! I, 413, 415, 417,465; CDRU SEQ ID NO: 47. 48, 49. 50. 51, 52, 53, 54. 55. 56, 57. 58, 60, 62, 64. 65, 67, 69, 71, 72, 74. 76. 77. 78. 79, 80, 8!, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 463; CDRL SEQ ID NO: 5. 7. 9, 10, 12, 13, 15. 16. US20120020976A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33. 35. 36, 37, 38, 39, 40, 42, 44, 46, 222, 229, 238, 405, 407, 409, 411, 413, 415, 417; CDRU SEQ ID NO: 47. 48, 49. 50. 51, 52, 53, 54. 55.56.57. 58, 60, 62, 64, 65, 67, 69, 71, 72, 74. 76. 77. 78. 79, 80, 8!, 83, 85, 87, 89, 91, 247, 256,265, 404, 406, 408,410, 412,414,416; CDRL SEQ ID NO: 5.7.9, 10. 12. 13. IS. 16. US20130085265A! 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33. 35. 36, 37. 38, 39, 40, 42, 44. 46, 405. 407, 409, 411, 413, 415, 417, 461, 465, 485; CDRH SEQ ID NO: 47. 48, 49, 50, 51, 52, 53, 54. 55. 56. 57. 58, 60, 62, 64, 65. 67, 69, 71, 72, 74. 76, 77. 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406,408, 410, 412, 414,416, 459.463,483; SEQ ID NOs comprising an anti-PCSK9 Patent or patent Pblication monoclonal antibody or fragment thereof CDRL SEQ ID NO: 5,7,9, 10, 12, 13, 15. 16. LJS20130079501A1 17, 18, 19,20, 21, 22, 23,24, 26, 28. 30, 31, 32, 33. 35. 36, 37. 38, 39, 40,42, 44. 46, 405, 407, 409, 411, 413, 415. 417, 461, 465, 485; CDR}I SEQ ID NO: 47. 48, 49. 50. 51, 52, 53, 54. 55. 56. 57. 58, 60, 62, 64, 65. 67, 69, 71, 72, 74. 76. 77. 78. 79, 80, 8!, 83. 85. 87, 89, 91, 404, 406,408. 410, 412, 414,416, 459,463,483; CDRL SEQ ID NO: 5,7,9, 10, 12, 13, IS, 16. US201202!3797A! 17, 18, 19,20, 21, 22, 23,24, 26, 28. 30, 31, 32, 33. 35. 36, 37. 38, 39, 40, 42, 44. 46, 405, 407, 409,411,413,415,417,158,162,395,473,477; CDRU SEQ ID NO: 47. 48, 49. 50. 51, 52, 53, 54. 55. 56, 57. 58, 60, 62, 64, 65, 67, 69, 71, 72, 74. 76, 77. 78, 79, 80, 8!, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 180. 175, 308, 368, 47!, 475; CDRL SEQ ID NO: 5.7,9, 10. 12. 13, 15, 16, US20120251544A1 17, 18, 19,20, 21, 22, 23,24, 26, 28, 30, 31, 32, 33. 35. 36, 37. 38, 39, 40, 42, 44. 46, 405. 407, 409,411, 413, 415. 417; CDRH SEQ ID NO: 47. 48, 49. 50. 51, 52, 53, 54. 55. 56. 57. 58, 60, 62, 64, 65. 67, 69, 71, 72, 74. 76, 77. 78, 79, 80, 8!, 83, 85, 87, 89, 91, 404, 406,408, 410, 412, 414,416; SEQ ID NOs comprising an anti-PCSK9 Patent or patent Pblication monoclonal antibody or fragment thereof CDRL SEQ ID NO: 5.7,9, 10, 12, 13, 15. 16. LJS20130052201A1 17, 18, 19,20, 21, 22, 23,24, 26, 28. 30, 31, 32, 33. 35. 36, 37. 38, 39, 40,42, 44. 46, 405, 407, 409, 411, 413, 415. 417, 461, 465, 485; CDR}I SEQ ID NO: 47. 48, 49. 50. 51, 52, 53, 54. 55. 56. 57. 58, 60, 62, 64, 65. 67, 69, 71, 72, 74. 76. 77. 78. 79, 80, 8!, 83. 85. 87, 89, 91, 404, 406,408. 410, 412, 414,416, 459,463,483; CDRL SEQ ID NO: 5.7,9, 10, 12, 13, IS, 16. US20130058944A! 17, 18, 19,20, 21, 22, 23,24, 26, 28. 30, 31, 32, 33. 35. 36, 37. 38, 39, 40, 42, 44. 46, 405, 407, 409,411,413,415,417,461,465,485; CDRU SEQ ID NO: 47. 48, 49. 50. 51, 52, 53, 54. 55. 56, 57. 58, 60, 62, 64. 65, 67, 69, 71, 72, 74. 76. 77. 78. 79, 80, 8!, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 459. 463, 483; CDRL SEQ ID NO: 5. 7. 9, 10, 12, 13, 15. 16. US20130079502A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33. 35. 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409,411. 413. 415. 417; CDRU SEQ ID NO: 47. 48, 49. 50. 51, 52, 53, 54. 55.56.57. 58, 60, 62, 64, 65, 67, 69, 71, 72, 74. 76. 77. 78. 79, 80, 8!, 83, 85, 87, 89, 91, 404, 406,408, 410, 412, 414.416; CDRL SEQ ID NO: 5.7.9, 10. 12. 13. IS. 16. US20130245235A! 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33. 35. 36, 37. 38, 39, 40, 42, 44. 46, 405. 407, 409,411. 413, 415. 417; CDRH SEQ ID NO: 47. 48, 49, 50, 51, 52, 53, 54. 55. 56. 57. 58, 60, 62, 64, 65. 67, 69, 71, 72, 74. 76, 77. 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406,408, 410, 412, 414,416;

Claims

CLAIMS: 1. A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOl), wherein the T01 is present in humans as different polymorphic variants, the method comprising a. Administering to the human an anti-TOl ligand to target a TOl variant in the human and treat or prevent said disease or condition, wherein the 101 in said human is encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or wherein the TOl in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%; wherein b. Before step (a) said human has been or is genotyped as positive for said nucleotide sequence or phenotyped as positive for said TOl variant.
2. The method of claim 1, wherein before step (a) the ligand has been or is determined as being capable of binding to said TOl variant.
3. A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOl), wherein the 101 is present in humans as different polymorphic variants, the method comprising a. Administering to the human an anti-TOl ligand to target a TOl variant in the human and treat or prevent said disease or condition, wherein the 101 in said human is encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or wherein the TOl in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%; wherein b. Before step (a) the ligand has been or is determined as capable of binding to said TOl variant.
4. The method of claim 3, wherein the genome of said human comprises a nucleotide sequence encoding said TOl variant; and before step (a) said nucleotide sequence has been or is determined as having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.
5. The method of claim 3 or 4, wherein said human has been or is genotyped as positive for said variant nucleotide sequence before step (a).
6. The method of any preceding claim, wherein the human has been or is phenotyped as positive for said TOl variant before step (a).
7. The method of any preceding claim, wherein said frequency is less than 10 or 15%.
8. The method of any preceding claim, wherein the ligand is capable of binding to two or more different TOl variants, each being encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.
9. A method of treating or preventing a disease or condition in a human, wherein the disease or condition is mediated by a Target of Interest (TOl), wherein the TOl is present in humans as different polymorphic variants, the method comprising a. Administering to the human an anti-TOl ligand to target a TOI variant in the human and treat or prevent said disease or condition, wherein the TOI in said human is a variant encoded by a nucleotide sequence having a cumulative human allele frequency of more than 50% and/or having a total human genotype frequency of more than 50%; wherein b. Before step (a) said human has been or is genotyped as negative for a variant nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; or phenotyped as negative for a TOl variant encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.
10. The method of claim 9, wherein before step (a), the human has been or is phenotyped as positive for the most frequent TOl variant or genotyped for the nucleotide sequence thereof.
11. The method of claim 9 or 10, wherein before step (a) the ligand has been or is determined as being capable of binding to the most frequent TOl variant.
12. The method of claim 9, 10 or 11, wherein before step (a) the ligand has been or is determined as being substantially incapable of neutralising or inhibiting said TOl variant recited in step (b).
13. The method of any one of claims 9 to 12, wherein the ligand is capable of binding to the most frequent TOl variant.
14. The method of any one of claims 9 to 13, wherein the ligand is capable of binding to two or more different TOl variants, each being encoded by a nucleotide sequence having a cumulative human allele frequency of more than 50%.
15. The method of any preceding claim, wherein said variant nucleotide sequence recited in step (a) has been or is determined as being present in at least 2 different human ethnic populations.
16. The method of any preceding claim, wherein said human frequency is the frequency in a database of naturally-occurring sequences sampled from at least 15 different human ethnic populations and comprising at least 1000 sequences.
17. An anti-human TOl ligand for use in a method of treating and/or preventing a TOl-mediated disease or condition in a human, wherein the TOl is present in humans as different polymorphic variants and wherein the genome of said human comprises a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, the method comprising administering the ligand to the human.
18. The ligand of claim 17, wherein the ligand has been or is determined as capable of binding the human TOl encoded by said nucleotide sequence.
19. A ligand that binds a human TOl comprising an amino acid sequence encoded by a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, for use in a method comprising the step of using the ligand to target said TOl in a human to treat and/or prevent a disease or condition mediated by TOl, the method comprising administering the ligand to the human.
20. The ligand of any one of claims 17 to 19, wherein the human has been or is genotyped as positive for said TOl nucleotide sequence having a cumulative human allele frequency of less than 50%.The ligand of any one of claims 17 to 19. wherein the human has been or is genotyped as positive for said TOl nucleotide sequence having a total human genotype frequency of less than 50%.
21. The ligand of any one of claims 17 to 20, wherein the human has been or is phenotyped as positive for a TOl encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.
22. The ligand of any one of claims 17 to 21, wherein the human has been or is genotyped as heterozygous for a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; optionally wherein the human has been or is genotyped as comprising a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and a TOl nucleotide sequence having a cumulative human allele frequency of more than 50% and/or having a total human genotype frequency of more than 50%.
23. The ligand of any one of claims 17 to 22, wherein the genome of the human has been or is genotyped as homozygous for a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.
24. The ligand of any one of claims 17 to 23, wherein the ligand comprises an antibody binding site that binds a human TOl comprising an amino acid sequence encoded by a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%; and optionally has been or is determined as capable of such binding.
25. The ligand of claim 24, wherein the ligand is an antibody or antibody fragment.
26. The ligand of any one of claims 17 to 23, wherein the ligand comprises a nucleotide sequence that specifically hybridises to a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50% or an RNA transcript thereof; and/or the ligand comprises a nucleotide sequence that comprises at least 10 contiguous nucleotides of a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50% or is an antisense sequence thereof.
27. The ligand of any one of claims 17 to 26, wherein the genome of said human comprises a nucleotide sequence having a cumulative human allele frequency of less than 50% and the sequence is found in at least 2 different ethnic populations.
28. A pharmaceutical composition or kit for treating or preventing a condition or disease mediated by a TOl as recited in any preceding claim, the composition or kit comprising a ligand of any one of claims 17 to 27; and optionally in combination with a label or instructions for use to treat and/or prevent said disease or condition in a human; optionally wherein the label or instructions comprise a marketing authorisation number (eg, an FDA or EMA authorisation number); optionally wherein the kit comprises an injection pen or IV container that comprises the ligand.
29. A method of producing an anti-human TOl antibody binding site, the method comprising obtaining a plurality of anti-TOl antibody binding sites, screening the antibody binding sites for binding to a TOl comprising an amino acid sequence encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, or to a peptide thereof that comprises an amino acid variation from the corresponding sequence encoded by the TOl-encoding nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency, and isolating an antibody binding site that binds in the screening step.
30. A method of producing an anti-human TOl antibody) the method comprising immunising a non-human vertebrate (eg, a mouse or a rat) with a TOl comprising an amino acid sequence encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, or to a peptide thereof that comprises an amino acid variation from the corresponding sequence encoded by the TOl-encoding nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency, and isolating an antibody that binds a TOl comprising an amino acid sequence encoded by a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, and optionally producing a TOl-binding fragment or derivative of the isolated antibody.
31. The method of claim 29 or 30, comprising the step of obtaining a nucleic acid encoding the antibody, fragment, derivative or binding site and optionally inserting the nucleic acid in an expression vector.
32. A kit for TOl genotyping a human, wherein the kit comprises a nucleic acid comprising a nucleotide sequence that specifically hybridises to a TOl nucleotide sequence selected having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or an RNA transcript thereof; and/or the nucleic acid comprises a nucleotide sequence that comprises at least 10 contiguous nucleotides of a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or is an antisense sequence thereof.
33. A kit for TOl genotyping or phenotyping a human, wherein the kit comprises a ligand according to any one of claims 17 to 27 or an antibody, fragment or derivative produced by the method of any one of claims 29 to 31.
34. Use of an anti-TOl ligand that binds a human TOl comprising an amino acid sequence encoded by a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, in the manufacture of a medicament for treating and/or preventing a TOl-mediated disease or condition in a human whose genome comprises a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.
35. Use of an anti-TOl ligand that binds a human TOl comprising an amino acid sequence encoded by a 101 nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, in the manufacture of a medicament for targeting said TOl in a human to treat and/or prevent a disease or condition mediated by 101.
36. A method of targeting a TOl for treating and/or preventing a TOl-mediated disease or condition in a human, the method comprising administering an anti-TOl ligand to a human comprising a 101 nucleotide sequence selected having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%, whereby a 101 encoded by said nucleotide sequence is targeted.
37. The method of claim 36, wherein the method comprises targeting a human TOI comprising an amino acid sequence with said ligand to treat and/or prevent said disease or condition in said human, wherein said amino acid sequence is encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50%.
38. A method of 101 genotyping a nucleic acid sample of a human, the method comprising identifying in the sample the presence of a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.
39. A method of 101 typing a protein sample of a human, the method comprising identifying in the sample the presence of a 101 amino acid sequence encoded by a 101 nucleotide sequence having a cumulative human allele frequency of less than 50% and/or having a total human genotype frequency of less than 50%.
40. The method of claim 38 or 39, comprising obtaining a sample of serum, blood, faeces) hair) urine or saliva from a human, whereby the nucleic acid or protein sample is obtained for use in the step of identifying said sequence.
41. The method of any one of claims 38 to 40, comprising using a ligand according to any one of claims 17 to 27 to carry out said identifying step.
42. A diagnostic kit comprising a ligand that is capable of binding a human TOl comprising an amino acid sequence encoded by a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% and instructions for carrying out the method of claim 38 or 39.
43. A diagnostic kit comprising a nucleic acid probe comprising a nucleotide sequence that specifically hybridises a TOl nucleotide sequence having a cumulative human allele frequency of less than 50% and/or a total human genotype frequency of less than 50% or an RNA transcript thereof and instructions for carrying out the method of claim 38 or 39.
44. The method, ligand, composition, kit or use of any preceding claim, wherein the TOl is encoded by a nucleotide sequence having a cumulative human allele frequency from ito 10% and/or a total human genotype frequency from ito about 15% or from 1 to 15%.
45. The method, ligand, composition, kit or use of any preceding claim wherein the TOl is a human TOl selected from Table 4; optionally for treating and/or preventing a corresponding disease or condition as set out in table 4.