Human anti- (poly-GA) dipeptide repeat (DPR) antibodies

文档序号：788711 发布日期：2021-04-09 浏览：18次中文

阅读说明：本技术 人源性抗(聚-ga)二肽重复序列(dpr)抗体 (Human anti- (poly-GA) dipeptide repeat (DPR) antibodies ) 是由 J.格里姆 F.蒙特拉西奥 I.达尔基利克-利德尔 M.M.拉什 J.W.阿恩特于 2019-04-25 设计创作，主要内容包括：提供了能够结合C9orf72聚甘氨酸-丙氨酸DPR的新型二肽重复序列(DPR)特异性抗体(例如,人源性抗体)以及其合成变体和生物技术衍生物；以及与所述抗体相关的方法和用途。本发明的抗体能够用于靶向DPR蛋白的免疫疗法和诊断剂的药物组合物和诊断组合物中。(Novel dipeptide repeat sequence (DPR) -specific antibodies (e.g., humanized antibodies) capable of binding C9orf72 polyglycine-alanine DPR are provided, as well as synthetic variants and biotechnological derivatives thereof; and methods and uses relating to the antibodies. The antibodies of the invention can be used in pharmaceutical and diagnostic compositions of immunotherapeutics and diagnostic agents targeting DPR proteins.)

1. A polypeptide having at least 6 repeats (GA) capable of binding to gene translation from, for example, chromosome 9 open reading frame 72(C9orf72)₆(SEQ ID NO: 80) an antibody to the dipeptide repeat (DPR) of poly (glycine-alanine) (GA), or a DPR-binding fragment thereof, wherein said antibody or DPR-binding fragment thereof comprises the following six Complementarity Determining Regions (CDRs) in its variable region:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 4 or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(c) a VH-CDR3, said VH-CDR3 comprising SEQ ID NO: 5 or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(d) A VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8, or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(e) a VL-CDR2, said VL-CDR2 comprising SEQ ID NO: 9 or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10 or a variant thereof, wherein said variant comprises one or two amino acid substitutions, optionally wherein said antibody is a human antibody, optionally wherein said antibody is a monoclonal antibody, optionally wherein said antibody is a human monoclonal antibody.

2. The antibody or DPR-binding fragment thereof of claim 1, comprising in its variable region

(a) Comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof (V)_H) A chain, wherein the variant comprises one or more amino acid substitutions; and

(b) comprises the amino acid sequence of SEQ ID NO: 7 or a variant thereof (V)_L) A chain, wherein the variant comprises one or more amino acid substitutions; optionally wherein

The V is_HAnd V_LThe chain amino acid sequences are respectively identical to SEQ ID NO: 2 and 7 are at least 90% identical.

3. The antibody or DPR-binding fragment thereof of claim 1 or 2, wherein

(a) The CDR does not contain asparagine (N) and/or glutamine (Q) which are easy to deamidate; and/or

(b) The V is_HAnd/or V_LThe chain amino acid sequence does not contain occupied glycosylation sites.

4. The antibody or DPR-binding fragment thereof of any one of claims 1 to 3, wherein the one, two or more amino acid substitutions are selected from

(a) Replacing asparagine (N) or glutamine (Q) that is susceptible to deamidation with a non-susceptible amino acid;

(b) replacing a small flexible amino acid directly adjacent to a readily deamidated N or Q with a larger amino acid, optionally wherein the adjacent amino acid is glycine (G);

(c) substituting at least one amino acid that results in removal of a glycosylation site, optionally wherein the at least one amino acid is within a glycosylation motif NXS or NXT; and/or

(d) One or more amino acids are substituted, which are conservative amino acid substitutions,

optionally, wherein said one or more amino acid substitutions of (a) and (b) are present in VH-CDR2, and said one or more amino acid substitutions of (c) are present in said V_LIn the chain, wherein

(i) In VH-CDR2, the amino acid sequence corresponding to SEQ ID NO: 2 and/or an asparagine (N) at position 54 corresponding to SEQ ID NO: 2 with another amino acid, optionally wherein the asparagine (N) is substituted with serine (S) or threonine (T), and/or wherein the glycine (G) is substituted with serine (S) or threonine (T); and/or

(ii) At the V_LIn the chain, the amino acid sequence corresponding to SEQ ID NO: 7 with another amino acid, optionally wherein the asparagine (N) is substituted with aspartic acid (D).

5. The antibody or DPR-binding fragment thereof of claim 1, wherein the antibody or DPR-binding fragment thereof comprises the following six CDRs in its variable region:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof, wherein,

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof,

(d) a VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,

(e) a VL-CDR2, said VL-CDR2 comprising SEQ ID NO: 9, and

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10.

6. The antibody or DPR-binding fragment thereof of claim 1, wherein the antibody or DPR-binding fragment thereof comprises the following six CDRs in its variable region:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 78 with a sequence of amino acids of seq id no,

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof,

(d) A VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,

(e) a VL-CDR2, said VL-CDR2 comprising SEQ ID NO: 9, and

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10.

7. The antibody or DPR-binding fragment thereof of claim 5 or 6, comprising in its variable region a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 of the amino acid sequence V_HAnd (3) a chain.

8. The antibody or DPR-binding fragment thereof of claim 5 or 6, comprising in its variable region a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24 of the amino acid sequence V_LAnd (3) a chain.

9. The antibody or DPR-binding fragment thereof of claim 5 or 6, comprising in its variable region a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 of the amino acid sequence V_HAnd a nucleic acid comprising SEQ ID NO: 24 of the amino acid sequence V_LAnd (3) a chain.

10. The antibody or DPR-binding fragment thereof of any one of claims 1 to 9, wherein

(i) Such as a cartoonThe antibody or DPR binding fragment pair poly- (GA), as determined by Surface Plasmon Resonance (SPR)₈The peptide (SEQ ID NO: 81) has a binding affinity corresponding to K_D(dissociation constant) less than 30nM, where K_a(association rate) less than 5x10 ⁵M^-1s^-1And K is_d(off-rate) less than 10x10^-3s^-1Optionally wherein the DPR binding fragment has a binding affinity corresponding to K as determined by Surface Plasmon Resonance (SPR)_D(dissociation constant) is 10nM to 30nM, where K_a(association rate) 1 to 5x10⁵M^-1s^-1And K is_d(dissociation rate) 2.5 to 10x10^-3s^-1(ii) a And/or

(ii) Fab fragments thereof have a thermal stability and melting point T, respectively, in the range of 78-82 deg.C, optionally about 79-81 deg.C, as determined by differential scanning calorimetry (VP-DSC)_m。

11. The antibody or DPR-binding fragment thereof of any one of claims 1 to 10, further comprising a CDR or V with which the antibody or DPR-binding fragment thereof binds_HAnd V_LChain amino acid sequence optionally a heterologous polypeptide sequence, optionally wherein the heterologous polypeptide sequence comprises a human constant domain optionally belonging to the IgG class, most optionally belonging to the IgG1 class or isotype.

12. The antibody or DPR-binding fragment thereof of any one of claims 1 to 11, which antibody or DPR-binding fragment thereof

(i) Binding to the poly-GA peptide only if the number n of repeats is ≦ 6;

(ii) identifying poly- (GA)₁₅Linear and conformational epitopes on the peptide (SEQ ID NO: 66);

(iii) Binds to a poly-GA peptide coupled to a BSA carrier protein with substantially the same affinity as the corresponding hydrophobically coated peptide;

(iv) substantially no or minimal cross-reactivity with unrelated amyloidogenic proteins; and/or

(v) A DPR-containing protein or an aggregated form thereof capable of binding to translation from the C9orf72 gene in the granular cell layer of the cerebellum of a C9orf72-FTLD patient.

13. The antibody of any one of claims 1 to 12, selected from the group consisting of: single-chain Fv fragment (scFv), F (ab ') fragment, F (ab) fragment and F (ab')₂Fragments, and/or the antibodies are chimeric murine-human antibodies or murine-derived antibodies.

14. One or more polynucleotides encoding the antibody or DPR-binding fragment thereof of any one of claims 1 to 13 or immunoglobulin V thereof_HOr V_LA strand, optionally wherein the polynucleotide is a cDNA and/or is operably linked to a heterologous nucleic acid.

15. One or more vectors comprising the polynucleotide of claim 14.

16. A host cell comprising the polynucleotide of claim 14 or the vector of claim 15.

17. A method for preparing an anti-DPR antibody or an immunoglobulin chain thereof, the method comprising

(a) Culturing the cell of claim 16; and

(b) isolating the antibody or immunoglobulin chain thereof from the culture.

18. An antibody or DPR-binding fragment thereof or immunoglobulin chain encoded by the polynucleotide of claim 14 or obtainable by the method of claim 17, optionally wherein the antibody or DPR-binding fragment thereof or the antibody or DPR-binding fragment thereof of any one of claims 1 to 13

(i) Detectably labeled with a label selected from the group consisting of: enzymes, radioisotopes, fluorophores, tags, labels, and heavy metals; or

(ii) Is linked to a drug.

19. A composition comprising the antibody or DPR-binding fragment thereof of any one of claims 1 to 13 or 18, the polynucleotide of claim 14, the vector of claim 15, or the cell of claim 16, optionally wherein the composition is

(i) A pharmaceutical composition, and further comprising a pharmaceutically acceptable carrier, optionally wherein the composition is a vaccine; or

(ii) The diagnostic composition or kit, optionally further comprises reagents conventionally used in immune-based diagnostic methods.

20. The antibody or DPR-binding fragment thereof of any one of claims 1 to 13 or 18, the polynucleotide of claim 14, the vector of claim 15 or the cell of claim 16 for use in the prophylactic treatment of a disease associated with or caused by a DPR-containing protein or aggregate form thereof, optionally wherein the disease is selected from the group consisting of: amyotrophic Lateral Sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and FTLD-ALS.

21. The antibody or DPR-binding fragment thereof of any one of claims 1 to 13 or 18, the polynucleotide of claim 14, the vector of claim 15 or the cell of claim 16 for use in the therapeutic treatment of a disease associated with or caused by a DPR-containing protein or aggregate form thereof, optionally wherein the disease is selected from the group consisting of: amyotrophic Lateral Sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and FTLD-ALS.

22. The antibody or DPR-binding fragment thereof of any one of claims 1 to 13 or 18, the polynucleotide of claim 14, the vector of claim 15 or the cell of claim 16 for use in the in vivo detection of a poly-GA DPR protein, e.g. an aggregated poly-GA DPR protein, or targeting a therapeutic and/or diagnostic agent against a poly-GA DPR protein, e.g. an aggregated poly-GA DPR protein, in a human or animal.

Technical Field

The present invention relates generally to antibody-based therapies and diagnostic methods. In particular, the present invention relates to novel antibodies of human origin and fragments, derivatives and biotechnological variants thereof that specifically bind to unconventional non-ATG translations, in particular to a poly-glycine-alanine (poly-GA) dipeptide repeat (DPR) -forming hexanucleotide repeat sequence as found in chromosome 9 open reading frame 72(C9orf72) and antigens comprising such DPR, which are useful for the treatment and diagnosis of diseases and disorders induced by aggregated DPR and DPR-containing proteins, respectively. Furthermore, the present invention relates to pharmaceutical and diagnostic compositions comprising said antibodies and variants and derivatives thereof, which are valuable both as diagnostic tools to identify diseases associated with DPR or aggregates thereof, such as frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), FTLD-ALS, and spinocerebellar ataxia type 36, and also as passive vaccination strategies for treating such diseases.

Background

Frontotemporal lobar degeneration (FTLD) belongs to a group of clinical, pathological and genetic heterogeneous disorders associated with atrophy of the frontal and temporal lobes of the brain. It is the second most common cause of early onset dementia. Cognitive symptoms are variable and include dementia, behavioral changes, and changes in personality, language dysfunction, and/or psychosis due to degeneration of the frontal and temporal cortex. Due to its symptoms, FTLD can be divided into three groups: (i) behavioral variant frontotemporal dementia (bvFTLD), (ii) Semantic Dementia (SD), or (iii) progressive non-fluent aphasia (PNFA). Patients with FTLD die 5-10 years after the onset of symptoms because there is no appropriate treatment. However, it has been shown that 50% of FTLD patients have a positive family history and appear to represent a continuum of disease with a common underlying pathogenesis compared to Amyotrophic Lateral Sclerosis (ALS). Although both autosomal dominant disorders are shown to be heterogeneous both genetically and pathologically, see, e.g., Vance et al, Brain 129(2006), 868-876, genetic analysis identified heterozygous amplified hexanucleotide repeats (GGGGCC) located between non-coding exons 1a and 1b of the C9orf72 gene as the most common genetic cause of FTLD and ALS; see, for example, DeJesus-Hernandez et al, Neuron 72(2011), 245-. In particular, it was shown that non-conventional non-ATG translation of sense transcripts in three alternating reading frames (i.e., amplified hexanucleotide repeats) resulted in the production, and aggregation of three different polypeptides, each consisting of two amino acid repeats (dipeptide repeats, DPR), i.e., poly- (Gly-Ala; GA), poly- (Gly-Pro; GP), and poly- (Gly-Arg; GR). Furthermore, translation of the corresponding antisense transcript leads to the production of poly- (Pro-Arg; PR), poly- (Pro-Ala; PA) and poly- (Gly-Pro; GP). These C9orf 72-dipeptide repeat (DPR) amplifications were shown to result in up to 30% of FTLD patients, 50% of ALS patients, and 80% of FTLD-ALS patients, with the highest mutation frequencies observed in the us and european union caucasian populations. Furthermore, patients with C9orf72-DPR amplifications with more than 19 repeat sequences had a lower age of onset, increased incidence of neurological disorders and were prone to psychosis or hallucinations compared to patients with other forms of FTLD and/or ALS; see, e.g., Harms et al, Neurobiol. aging 34(2013), e13-e 19.

The deletions are useful in the treatment of diseases and/or disorders associated with dipeptide repeat sequence (DPR) amplification, e.g., drugs that slow disease progression. To date, the main focus of medical care has been to provide medications to treat the often very stressful accompanying symptoms.

The most recent approach in the therapeutic targeting of C9orf72 pathological amplifications against ALS, FTLD and other neurodegenerative diseases has focused on antisense oligonucleotide/RNA interference (RNAi) strategies using small molecule compounds to counteract the toxic effects directly exerted by RNA derived from repeat sequence transcription (foci), translation of dipeptide repeat sequence protein (DPR) from repeat sequences, or sequestration of RNA binding protein from C9orf72 amplification; and gene therapy, not only for silencing toxic RNA/proteins, but also for remedying single-dose insufficiency due to reduced transcription of the C9orf72 coding sequence or due to reduced availability of the RNA binding protein to be sequestered by the RNA lesion; for a review see, e.g., misc et al, mol. neurobiol.54(2017), 4466-.

Although it might be attractive to consider antibodies targeting DPR protein in the brain of patients with amplification of the hexanucleotide repeat of C9orf72, to date it appears that this approach has not been properly considered or may be influenced by the actual approach employed. For example, the group of Edbauer et al describes the synthesis of a protein by aggregation (GA) ₁₀Peptide (SEQ ID NO: 72) immunization of mice to generate antibodies against oligopeptides consisting of the dipeptide repeat sequence of sequence (Gly-Ala), the aggregated (GA)₁₀Peptide display and peptide display containing GA-DPR (GA)₁₅(SEQ ID NO: 66) GST-fusion protein binding; see international application WO 2014/114660. One of those antibodies (designated GA-5F2) could be shown to inhibit the spread and aggregation of C9orf72 poly-GA dipeptide repeat protein in vitro co-culture assays and cell extracts; see Zhou et al EMBO Molecular Medicine 9(2017), 687-.

However, in addition to the disadvantage that mouse monoclonal antibodies are liable to elicit human anti-mouse antibody (HAMA) responses in humans, DPR-GA has not been clarified since monoclonal antibodies have been raised against artificial antigens₁₅Whether or not the binding translates into a corresponding specificity and affinity for the DPR protein from the C9orf72 gene present in the brain of patients with ALS, FTLD and other neurodegenerative disorders.

Indeed, it appears that the academia and industry still seem to focus on RNA-targeted therapeutic strategies against C9orf72 ALS/FTLD; see, for example, Simone et al, reviews of recent releases of EMBO Molecular Medicine 10(2018), 22-31 and Schludi and Edbauer, EMBO Molecular Medicine 10(2018), 4-6, which report encouraging results of targeting RNA G-quadruplexes for improving C9orf72ALS/FTLD pathology in vivo and in vitro in Drosophila expressing GGGGCC repeats.

However, RNA-based therapeutics, small organic compounds and gene therapy suffer from several drawbacks, such as the inherent instability of RNA, the potentially immunogenic nature of the compounds and the need for delivery vehicles that are effectively transported to the target cells, and ethical issues with respect to the use of gene therapy still exist.

Accordingly, there remains a need to develop new drugs that therapeutically target the pathological expansion of C9orf72 in the treatment of ALS, FTLD, and other neurodegenerative disorders that are specific for diseases and disorders caused by the expression product of the C9orf72 gene, and optionally belong to a well-studied class of drugs, and are tolerable in humans.

This technical problem is solved by the embodiments characterized in the claims and further described below and shown in the examples and figures.

Disclosure of Invention

The present invention provides human monoclonal antibodies that are particularly useful in preventing or treating diseases and conditions associated with DPR proteins and aggregated forms thereof, which are capable of binding to dipeptide repeats (DPR) and DPR-containing proteins consisting of poly-glycine-alanine (Gly-Ala; GA) repeats (DPR proteins), as well as equivalent DPR protein binding molecules such as the DPR binding fragments, synthetic variants of antibodies and biotechnological derivatives exemplified herein.

Recently, a class of humanized anti-DPR antibodies has been described that hold promise for the development of antibody-based therapeutic interventions for the treatment of C9orf72-ALS and FTLD patients; see international application WO 2016/050822, the disclosure of which is incorporated herein by reference. As described therein, anti-DPR protein antibodies and cdnas encoding variable regions thereof have been isolated from patients with asymptomatic neurological and neurodegenerative disorders, respectively; see page 3 and examples of WO 2016/050822. In further experiments performed according to the present invention, an anti-poly-GA DPR antibody, designated NI-308.5J10 and hereinafter also referred to as "subject antibody" can be identified and cloned, which can be shown to have unique binding properties as determined in different binding assays, in particular on brain tissue from selected human C9orf72-FTLD patients; see example 11 and figure 10. In addition, as shown in example 9 and FIG. 8, the subject antibodies were conjugated to aggregated C9orf72 poly-GA DPR (GA)₁₅Binding of (SEQ ID NO: 66) was not blocked by the binding of the reference anti-poly-GA antibody (NI-mAb reference) to the target, while binding of the reference anti-poly GA antibody to the C9orf72 DPR peptide was abolished by previous binding of the subject NI-308.5J10 antibody to the target.

Thus, although both the subject NI-308.5J10 antibody and the reference anti-poly GA antibody have selected and recognized the C9orf72 poly-GA DPR protein or aggregated form thereof, surprisingly, the subject antibody appears to recognize additional conformational epitopes on the poly-GA peptide. This property makes the antibody particularly suitable for targeting C9orf72 DPR protein in patients carrying C9orf72 hexanucleotide repeat expansion, as poly-GA DPR protein aggregates often co-aggregate with other DPR proteins and/or unrelated aggregate proteins such as p62 and hnRNP A3 (Mori et al, Acta Neuropathia.126 (2013), 881-893; Mann et al, Acta Neuropathia. Communications (2013), 1: 68. doi: 10.1186/2051-5960-1-68; Davidson et al, Acta Neuropathia Communications (2017); 5: 31. doi: 10.1186/s 40478-017-0437-5).

In addition, further experiments conducted within the scope of the present invention revealed that amino acids in the CDRs and framework regions that are susceptible to deamidation or glycosylation can be substituted without losing the basic binding properties of the subject antibodies; see examples 12 to 15 and figure 11.

Thus, the present invention also provides variants and derivatives of the original human-derived anti-DPR antibodies, which variants and derivatives contain one or more amino acid substitutions within the CDR and/or framework regions, which one or more amino acid substitutions result in improved manufacturability of the antibody, while the binding properties and stability of the antibody remain unaffected or even improved; see examples 15 and 16. In addition, the subject variants and derivatives of the original NI-308.5J10 antibody are also expected to be substantially non-immunogenic in humans due to the only minor modifications in the CDRs and/or variable regions.

In summary, as disclosed in the present application, the present invention provides a human anti-poly-GA antibody as well as variants and derivatives thereof having properties making it particularly suitable for targeting C9orf72 DPR protein or aggregated forms thereof in the human brain and thus for immunotherapy of C9orf72 ALS/FTLD patients.

Accordingly, the present invention generally relates to the following embodiments:

[1]a polypeptide having at least 6 repeats (GA) capable of binding to a protein translated from, for example, chromosome 9 open reading frame 72(C9orf72)₆(SEQ ID NO: 80) an antibody to the dipeptide repeat (DPR) of poly (glycine-alanine) (GA), or a DPR-binding fragment thereof, wherein said antibody or DPR-binding fragment thereof comprises the following six Complementarity Determining Regions (CDRs) in its variable region:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 (e.g., comprising SEQ ID NO: 78) or a variant thereof, wherein the variant comprises one or two amino acid substitutions,

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 4 or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(c) a VH-CDR3, said VH-CDR3 comprising SEQ ID NO: 5 or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(d) A VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8, or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(e) a VL-CDR2, said VL-CDR2 comprising SEQ ID NO: 9 or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10 or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

optionally, wherein the antibody is a human antibody, optionally wherein the antibody is a monoclonal antibody, optionally wherein the antibody is a human monoclonal antibody.

[2] The antibody or DPR-binding fragment thereof according to [1], which comprises in the variable region thereof

(a) Comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof (V)_H) A chain, wherein the variant comprises one or more amino acid substitutions; and

(b) comprises the amino acid sequence of SEQ ID NO: 7 or a variant thereof (V)_L) A chain, wherein the variant comprises one or more amino acid substitutions; optionally wherein

The V is_HAnd V_LThe chain amino acid sequences are respectively identical to SEQ ID NO: 2 and 7 are at least 90% identical.

[3] The antibody or DPR-binding fragment thereof according to [1] or [2], wherein

(a) The CDR does not contain asparagine (N) and/or glutamine (Q) which are easy to deamidate; and/or

(b) The V is_HAnd/or V_LThe chain amino acid sequence does not contain occupied glycosylation sites.

[4] The antibody or DPR-binding fragment thereof of any one of [1] to [3], wherein the one, two or more amino acid substitutions are selected from

(a) Replacing asparagine (N) or glutamine (Q) that is susceptible to deamidation with a non-susceptible amino acid;

(b) replacing a small flexible amino acid directly adjacent to a readily deamidated N or Q with a larger amino acid, optionally wherein the adjacent amino acid is glycine (G);

(c) substituting at least one amino acid that results in removal of a glycosylation site, optionally wherein the at least one amino acid is within a glycosylation motif NXS or NXT; and/or

(d) The substitution is one or more amino acids that are conservative amino acid substitutions.

[5]Such as [4]]The antibody or DPR-binding fragment thereof of (a), wherein the one or more amino acid substitutions of (a) and (b) are present in a VH-CDR2, and the one or more amino acid substitutions of (c) are present in the V_LIn the chain.

[6] The antibody or DPR-binding fragment thereof according to [5], wherein

(ii) At the V_LIn the chain, the amino acid sequence corresponding to SEQ ID NO: 7 with another amino acid, optionally wherein the asparagine (N) is substituted with aspartic acid (D).

[7]Such as [1 ]]To [6 ]]The antibody or DPR-binding fragment thereof of any one of, wherein the antibody or DPR-binding fragment is directed to poly- (GA) as determined by Surface Plasmon Resonance (SPR)₈The peptide (SEQ ID NO: 81) has a binding affinity corresponding to K_D(dissociation constant) less than 30nM, where K_a(association rate) less than 5x 10⁵M^-1s^-1And K is_d(dissociation rate) less than 10x 10^-3s^-1Optionally wherein the DPR binding fragment pair has a binding affinity corresponding to K as determined by Surface Plasmon Resonance (SPR)_D(dissociation constant) is 10nM to 30nM, where K_a(association rate) 1 to 5x 10⁵M^-1s^-1And K is_d(dissociation rate) of 2.5 to 10x 10^-3s^-1。

[8]Such as [1 ]]To [7 ]]The antibody or DPR-binding fragment thereof of any one of (a) wherein the Fab fragment thereof has a thermostability and a melting temperature T, respectively, in the range of 78 ℃ -82 ℃, e.g. in the range of about 79 ℃ -81 ℃, as determined by differential scanning calorimetry (VP-DSC) _m。

[9] The antibody or DPR-binding fragment thereof according to any one of [1] to [8], which comprises in its variable region

(i) The following six CDRs:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 (e.g., comprising SEQ ID NO: 78),

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof,

(d) a VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,

(e) a VL-CDR2, the VL-CDR2 comprising SEQ id no: 9, or a pharmaceutically acceptable salt thereof, wherein,

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10;

(ii) the following six CDRs:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 (e.g., comprising SEQ ID NO: 78),

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, wherein,

(d) a VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,

(e) a VL-CDR2, the VL-CDR2 comprising SEQ id no: 9, or a pharmaceutically acceptable salt thereof, wherein,

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10;

(iii) The following six CDRs:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 (e.g., comprising SEQ ID NO: 78),

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 19, or a pharmaceutically acceptable salt thereof, wherein,

(d) a VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,

(e) a VL-CDR2, the VL-CDR2 comprising SEQ id no: 9, or a pharmaceutically acceptable salt thereof, wherein,

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10;

(iv) the following six CDRs:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 (e.g., comprising SEQ ID NO: 78),

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 22, or a pharmaceutically acceptable salt thereof,

(d) a VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,

(e) a VL-CDR2, the VL-CDR2 comprising SEQ id no: 9, or a pharmaceutically acceptable salt thereof, wherein,

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10; or

(v) The following six CDRs:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 (e.g., comprising SEQ ID NO: 78),

(b) A VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 4 in the sequence of amino acids of (a),

(d) a VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,

(e) a VL-CDR2, the VL-CDR2 comprising SEQ id no: 9, or a pharmaceutically acceptable salt thereof, wherein,

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10.

[9a] The antibody or DPR-binding fragment thereof of any one of [1] to [8], comprising the following six CDRs in its variable region:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 (e.g., comprising SEQ ID NO: 78),

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof,

(d) a VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,

(e) a VL-CDR2, the VL-CDR2 comprising SEQ id no: 9, and

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10.

[10] The antibody or DPR-binding fragment thereof according to any one of [1] to [9a ], which comprises in its variable region

(i) As shown in SEQ ID NO: 12 and SEQ ID NO: v shown in 24_HAnd V_LA chain amino acid sequence;

(ii) as shown in SEQ ID NO: 2 and SEQ ID NO: v shown in 24_HAnd V_LA chain amino acid sequence;

(iii) as shown in SEQ ID NO: 15 and SEQ ID NO: v shown in 24_HAnd V_LA chain amino acid sequence;

(iv) as shown in SEQ ID NO: 18 and SEQ ID NO: v shown in 24_HAnd V_LA chain amino acid sequence;

(v) as shown in SEQ ID NO: 21 and SEQ ID NO: v shown in 24_HAnd V_LA chain amino acid sequence;

(vi) as shown in SEQ ID NO: 12 and SEQ ID NO: v shown in 7_HAnd V_LA chain amino acid sequence;

(vii) as shown in SEQ ID NO: 15 and SEQ ID NO: v shown in 7_HAnd V_LA chain amino acid sequence;

(viii) as shown in SEQ ID NO: 18 and SEQ ID NO: v shown in 7_HAnd V_LA chain amino acid sequence;

(ix) as shown in SEQ ID NO: 21 and SEQ ID NO: 7 toV of the show_HAnd V_LA chain amino acid sequence; or

(x) As shown in SEQ ID NO: 2 and SEQ ID NO: v shown in 7_HAnd V_LA chain amino acid sequence.

[10a]Such as [1 ]]To [10 ]]The antibody of any one of claims or a DPR-binding fragment thereof comprising a V in its variable region_HChain, said V_HThe strand comprises a sequence identical to SEQ ID NO: 12, an amino acid sequence which is at least 90% identical to the amino acid sequence of seq id no.

[10b]Such as [1 ]]To [10a ]]The antibody of any one of claims or a DPR-binding fragment thereof comprising a V in its variable region _LChain, said V_LThe strand comprises a sequence identical to SEQ ID NO: 24, an amino acid sequence that is at least 90% identical.

[10c]Such as [1 ]]To [10b ]]The antibody of any one of claims or a DPR-binding fragment thereof comprising a V in its variable region_HChain, said V_HThe strand comprises a sequence identical to SEQ ID NO: 12, an amino acid sequence at least 95% identical.

[10d]Such as [1 ]]To [10c ]]The antibody of any one of claims or a DPR-binding fragment thereof comprising a V in its variable region_LA chain comprising a sequence identical to SEQ ID NO: 24, an amino acid sequence that is at least 95% identical.

[10e]Such as [1 ]]To [10d ]]The antibody of any one of claims or a DPR-binding fragment thereof comprising a V in its variable region_HChain, said V_HThe strand comprises a sequence identical to SEQ ID NO: 12, an amino acid sequence which is at least 99% identical to the amino acid sequence of seq id no.

[10f]Such as [1 ]]To [10e]The antibody of any one of claims or a DPR-binding fragment thereof comprising a V in its variable region_LA chain comprising a sequence identical to SEQ ID NO: 24, an amino acid sequence that is at least 99% identical.

[10g]Such as [1 ]]To [10f]The antibody of any one of claims or a DPR-binding fragment thereof comprising a V in its variable region _HThe chain is provided with a plurality of chains,the V is_HThe strand comprises the sequence relative to SEQ ID NO: 12 has 1, 2 or 3 additions, substitutions or deletions of SEQ ID NO: 12.

[10h]Such as [1 ]]To [10g ]]The antibody of any one of claims or a DPR-binding fragment thereof comprising a V in its variable region_LA chain comprising a variable domain relative to SEQ ID NO: 24 has 1, 2 or 3 additions, substitutions or deletions of SEQ ID NO: 24.

[10i]Such as [1 ]]To [10h ]]The antibody of any one of claims or a DPR-binding fragment thereof comprising a V in its variable region_HA chain, said VH chain comprising SEQ ID NO: 12.

[10j]Such as [1 ]]To [10i]The antibody of any one of claims or a DPR-binding fragment thereof comprising a V in its variable region_LA chain comprising SEQ ID NO: 24.

[11]Such as [1 ]]To [10j ]]The antibody or DPR-binding fragment thereof of any one of, further comprising a CDR or V_HAnd V_LChain amino acid sequence optionally a heterologous polypeptide sequence, optionally wherein the heterologous polypeptide sequence comprises a human constant domain, optionally of the IgG type, optionally of the IgG1 class or isotype.

[11a] The antibody or DPR-binding fragment thereof of [11], wherein the heterologous polypeptide sequence is heterologous to the CDR.

[11b]Such as [11]]The antibody or DPR-binding fragment thereof, wherein the heterologous polypeptide sequence is associated with the V_HAnd V_LAre heterologous.

[11c] The antibody or DPR-binding fragment thereof of any one of [11] to [11b ], wherein the heterologous polypeptide sequence is a light chain constant domain, optionally of the kappa type.

[11d] The antibody or DPR-binding fragment thereof of any one of [11] to [11b ], wherein the heterologous polypeptide sequence is a heterologous mammalian secretion signal peptide.

[12] The antibody or DPR-binding fragment thereof of any one of [1] to [11d ], optionally, the antibody or DPR-binding fragment thereof binds to a poly-GA peptide only if the number of repeats, n ≧ 6.

[13]Such as [1]]To [12]]The antibody of any one of claims or a DPR-binding fragment thereof, wherein the antibody recognizes poly- (GA)₁₅Linear and conformational epitopes on the peptide (SEQ ID NO: 66).

[14]Such as [1]]To [13 ]]The antibody or DPR-binding fragment thereof of any one of (a) wherein the antibody binds to poly- (GA) with an affinity KD of about (0.05-0.5nM, optionally 0.1-0.2nM) as determined by biolayer interferometry ₁₅Peptide (SEQ ID NO: 66) with an association rate constant of (K)_a＝0.5-5x 10⁵M^-1s^-1) And a dissociation constant of (K)_d＝1-5x 10^-5s^-1)。

[15] The antibody or DPR-binding fragment thereof of any one of [1] to [14], wherein the antibody binds to a poly-GA peptide coupled to a BSA carrier protein with substantially the same affinity for the corresponding hydrophobically coated peptide.

[16] The antibody or DPR-binding fragment thereof of any one of [1] to [15], which is substantially free of, or minimally cross-reactive to, an unrelated amyloidogenic protein.

[17] The antibody or DPR-binding fragment thereof of any one of [1] to [16], wherein the antibody is capable of binding a DPR-containing protein or an aggregated form thereof as translated from the C9orf72 gene in the granular cell layer of the cerebellum of a C9orf72-FTLD patient.

[18] The antibody or DPR-binding fragment thereof of any one of [1] to [17], which is capable of ameliorating at least one symptom of a pathological hallmark of C9orf72 disease, such as neuronal loss, behavioral abnormalities, dyskinesias, and decreased survival, if administered to a transgenic C9orf72 mouse model.

[19]Such as [1] ]To [18 ]]The antibody of any one of the above, selected from the group consisting of: single-chain Fv plateSegment (scFv), F (ab ') fragment, F (ab) fragment and F (ab')₂And (3) fragment.

[20] The antibody according to any one of [1] to [19], which is a chimeric murine-human antibody or a murine-derived antibody.

[21]One or more polynucleotides encoding a polypeptide as described in [1]]To [20]]The antibody of any one of the above, or a DPR-binding fragment thereof, or an immunoglobulin V thereof_HOr V_LA strand, optionally wherein the polynucleotide is a cDNA and/or is operably linked to a heterologous nucleic acid.

[21a]A polynucleotide encoding a polypeptide as described in [1]]To [20]]V of the antibody of any one of the above or a DPR-binding fragment thereof_HA strand wherein when compared to a polypeptide comprising SEQ ID NO: 24 of the amino acid sequence V_LWhen the chains are paired, V_HThe strand binds to a DPR of a poly-GA having at least 6 repeats as translated from the C9orf72 gene, or a DPR-binding fragment thereof, optionally wherein the polynucleotide is a cDNA and/or is operably linked to a heterologous nucleic acid.

[21b]A polynucleotide encoding a polypeptide as described in [1]]To [20]]V of the antibody of any one of the above or a DPR-binding fragment thereof_LA strand wherein when compared to a polypeptide comprising SEQ ID NO: 12 of the amino acid sequence V _HWhen the chains are paired, V_LThe strand binds to a DPR of a poly-GA having at least 6 repeats as translated from the C9orf72 gene, or a DPR-binding fragment thereof, optionally wherein the polynucleotide is a cDNA and/or is operably linked to a heterologous nucleic acid.

[21c] The polynucleotide of any one of [21] to [21b ], wherein the heterologous nucleic acid is a regulatory element.

[21d] The polynucleotide of any one of [21] to [21b ], wherein the heterologous nucleic acid is a promoter, an enhancer, a ribosome binding site, or a transcription terminator, optionally wherein the promoter is a cytomegalovirus immediate early promoter.

[21e] The polynucleotide of any one of [21] to [21b ], wherein the heterologous nucleic acid encodes a secretion signal peptide, optionally wherein the secretion signal peptide is a mammalian signal peptide.

[22] One or more vectors comprising the polynucleotide of any one of [21] to [21e ].

[23] A host cell comprising the polynucleotide of any one of [21] to [21e ] or the vector of [22 ].

[24] Use of the polynucleotide of any one of [21] to [21e ], the vector of [22], or the host cell of [30] for the production of an anti-DPR antibody.

[25] A method for preparing an anti-DPR antibody or an immunoglobulin chain thereof, the method comprising

(a) Culturing the cell of [23 ]; and

(b) isolating the antibody or immunoglobulin chain thereof from the culture.

[26] An antibody or a DPR-binding fragment thereof or an immunoglobulin chain encoded by a polynucleotide as described in any one of [21] to [21e ] or obtainable by the method as described in [25] or the use as described in [24 ].

[27] The antibody or DPR-binding fragment thereof according to any one of [1] to [20] or [26], which is

(i) Detectably labeled with a label selected from the group consisting of: enzymes, radioisotopes, fluorophores, tags, labels, and heavy metals; or

(ii) Is linked to a drug.

[28] A composition comprising the antibody or DPR-binding fragment thereof of any one of [1] to [20], [26] or [27], the polynucleotide of any one of [21] to [21e ], or the vector of [22], or the cell of [23 ].

[29] The composition of [28], which is a pharmaceutical composition and further comprising a pharmaceutically acceptable carrier, optionally wherein the composition is a vaccine.

[30] A method of preparing a pharmaceutical composition for treating a condition associated with or caused by a DPR-containing protein or an aggregated form thereof, the method comprising:

(a) culturing the cell of [23 ];

(b) purifying the antibody or immunoglobulin chain thereof from the culture to pharmaceutical grade; and

[31] The composition of [28], which is a diagnostic composition or kit, optionally further comprising reagents conventionally used in immune-based diagnostic methods.

[32] The antibody or DPR-binding fragment thereof according to any one of [1] to [20], [26] or [27], the polynucleotide according to [21], the vector according to [22] or the cell according to [23], for use in the prophylactic treatment of a disease associated with or caused by a DPR-containing protein or an aggregated form thereof.

[32a] The antibody or DPR-binding fragment thereof according to any one of [1] to [20], [26] or [27], the polynucleotide according to [21], the vector according to [22] or the cell according to [23], for use in the therapeutic treatment of a disease associated with or caused by a DPR-containing protein or an aggregated form thereof.

[32b] The antibody or DPR-binding fragment thereof according to any one of [1] to [20], [26] or [27], the polynucleotide according to [21], the vector according to [22] or the cell according to [23], for use in the prophylactic and therapeutic treatment of a disease associated with or caused by a DPR-containing protein or an aggregate form thereof.

[33] An antibody or DPR-binding fragment thereof, a polynucleotide, a vector or a cell for the use according to any one of [32] to [32b ], wherein the disease is selected from the group consisting of: frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), and FTLD-ALS.

[34] An antibody or DPR-binding fragment thereof, polynucleotide, vector or cell for use according to any one of [32] to [32b ] and [33], wherein the antibody is capable of ameliorating at least one symptom of a pathological hallmark of C9orf72 disease, such as neuronal loss, behavioral abnormalities, dyskinesias and decreased survival when administered to a transgenic C9orf72 mouse model.

[35] The antibody or DPR-binding fragment thereof according to any one of [1] to [20], [26] or [27], for use in the in vivo detection of a poly-GA DPR protein, e.g., an aggregated poly-GA DPR protein, in the human or animal body, or a therapeutic and/or diagnostic agent targeted against a poly-GA DPR protein, e.g., an aggregated poly-GA DPR protein, in the human or animal body.

[36] The antibody or DPR binding fragment thereof for use according to [35], wherein the in vivo imaging comprises Positron Emission Tomography (PET), single photon emission tomography (SPECT), Near Infrared (NIR), optical imaging or Magnetic Resonance Imaging (MRI).

Further, provided herein are DPR Ab-1 antibodies or fragments thereof. Additionally, provided herein is an anti-DPR antibody or fragment thereof comprising a Complementarity Determining Region (CDR), a heavy chain sequence, a light chain sequence, a variable domain sequence, and/or a constant domain sequence set forth in table 12. In embodiments, the anti-DPR antibody or fragment thereof comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 38 and a light chain having the amino acid sequence of SEQ ID NO: 42, or a light chain of the amino acid sequence of seq id no.

Also provided herein is a nucleic acid molecule comprising:

(i) a nucleic acid sequence encoding a heavy chain of an anti-DPR antibody, said heavy chain having the amino acid sequence of SEQ ID NO: 38; and/or

(ii) A nucleic acid sequence encoding a light chain of an anti-DPR antibody, the light chain having the amino acid sequence of SEQ ID NO: 42, optionally wherein the nucleic acid sequences (i) and (ii) are located on the same nucleic acid molecule or on separate nucleic acid molecules,

Optionally wherein the nucleic acid molecule comprises cDNA and/or is operably linked to a heterologous nucleic acid.

Also provided herein is a nucleic acid molecule comprising SEQ ID NO: 51-58.

Also provided herein is a vector comprising a nucleic acid molecule described herein.

Also provided herein is a host cell comprising (i) a nucleic acid molecule described herein or (ii) a vector described herein.

In some aspects, provided herein is the use of a nucleic acid molecule, vector or host cell described herein for the production of an anti-DPR antibody or fragment thereof.

In some aspects, provided herein is a method of producing an anti-DPR antibody or fragment thereof, the method comprising: (i) culturing a host cell as described herein; and (ii) isolating the antibody or fragment thereof from the culture.

Also provided herein is a composition, e.g., a pharmaceutical composition, comprising an anti-DPR antibody or fragment thereof described herein, a nucleic acid molecule described herein, a vector described herein, or a host cell described herein. In embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In embodiments, the composition is a diagnostic composition or kit, e.g., further comprising reagents conventionally used in immune-based diagnostic methods.

Also provided is a method of treating a disease associated with or caused by a DPR-containing protein aggregate (e.g., a protein aggregate containing C9ORF72 DPR) in a subject in need thereof (e.g., Amyotrophic Lateral Sclerosis (ALS), frontotemporal lobar degeneration (FTLD), or FTLD-ALS), the method comprising administering to the subject an anti-DPR antibody or fragment thereof described herein (e.g., DPR Ab-1, e.g., containing an amino acid sequence set forth in table 12), thereby treating a disorder in the subject (e.g., ALS, FTLD, or FTLD-ALS).

Also provided is a method of making a pharmaceutical composition for treating a disorder associated with or caused by a DPR-containing protein or aggregated form thereof (e.g., ALS, FTLD, or FTLD-ALS), the method comprising: (i) culturing a host cell as described herein; (ii) isolating and/or purifying the antibody or fragment thereof from the culture that reaches pharmaceutical grade; and (iii) mixing the antibody or fragment thereof with a pharmaceutically acceptable carrier.

Provided herein is an anti-DPR antibody or fragment thereof described herein (e.g., DPRAb-1, e.g., comprising an amino acid sequence set forth in table 12), a nucleic acid molecule described herein, a vector described herein, or a host cell described herein for use in the treatment (e.g., prophylactic and/or therapeutic treatment) of a disorder associated with or caused by a DPR-containing protein or aggregated form thereof (e.g., ALS, FTLD, or FTLD-ALS).

Provided herein is the use of an anti-DPR antibody or fragment thereof described herein (e.g., DPR Ab-1, e.g., comprising an amino acid sequence set forth in table 12), a nucleic acid molecule described herein, a vector described herein, or a host cell described herein, for the manufacture of a medicament for the treatment (e.g., prophylactic and/or therapeutic treatment) of a disorder associated with or caused by a DPR-containing protein or aggregated form thereof (e.g., ALS, FTLD, or FTLD-ALS).

Other embodiments of the present invention will be apparent from the following description and examples.

Drawings

FIG. 1: the amino acid sequence of the variable region of human antibody NI-308.5J10 and variants thereof. Framework (FR) regions and Complementarity Determining Regions (CDRs) are shown, with the CDRs underlined. The Kabat numbering scheme was used (seehttp:// www.bioinf.org.uk/abs/；Kabat et al, u.s.depth.of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983), cited in the mentioned network references and given in table 1 at pages 39 and 40 of WO 2016/050822 a2, incorporated herein by reference. Unless otherwise specified, reference to numbering specific amino acid residue positions in an antibody of the invention, or a DPR-binding fragment, variant or derivative thereof, is according to the Kabat numbering system, however, this is theoretical and may not apply equally to each antibody of the invention. For example, depending on the position of the first CDR, subsequent CDRs may move in either direction. Thus, based on the disclosure herein, one skilled in the art would appreciate that in the event of any unexpected error or inconsistency with respect to the indications of the CDR and/or sequence listing in FIG. 1, i.e., variable weight (V) of antibody NI-308.5J10 _H) And variable lightness (V)_L) The chain amino acid sequence is suitably in accordance with Kabat determines the position of the correct CDR sequences, which applies to define the claimed antibodies and DPR-binding fragments thereof. (A) Variable heavy chain VH and light chain VK sequences of antibody NI-308.5J10, as shown in SEQ ID NO: 2 and 7; (B) the variable heavy chain sequence VH of antibody NI-308.5J10 variant with amino acid substitution N54S, as shown in SEQ ID NO: shown in fig. 12. Preferred amino acid substitutions within the CDRs of the variable heavy and light chain sequences are shown in bold, including those present in the variable heavy chain sequences shown in (c) to (e) and the variable light chain sequences shown in (f). As further explained in the specification, conservative amino acid substitutions are preferred within the CDR and/or framework regions, taking into account the physicochemical properties of the original amino acids, either alone or together with adjacent amino acids, as described by Mirsky et al, mol.biol.Evol.32(2014)806-819 at 813, FIG. 6, in particular the LG model,for exampleSuch that the positions of two amino acids are changed, e.g., "PS" and "SP" in VL-CDR3, which have been found in the variable light chain of human anti-polyGA antibodies with similar but non-identical binding properties, possibly due to substitutions of two or more amino acids in one or more of the other CDRs. (C) The variable heavy chain sequence VH of antibody NI-308.5J10 variant with amino acid substitution N54T, as shown in SEQ ID NO: 15, and (b); (D) the variable heavy chain sequence VH of antibody NI-308.5J10 variant with amino acid substitution G55S, as shown in SEQ ID NO: 18; (E) the variable heavy chain sequence VH of antibody NI-308.5J10 variant with amino acid substitution G55T, as shown in SEQ ID NO: 21, and (b); (F) variable light chain sequence VK of antibody NI-308.5J10 variant with amino acid substitution N75D, as shown in SEQ ID NO: shown in 24.

FIG. 2: binding specificity and EC for C9orf72 dipeptide repeat protein₅₀And (4) measuring. Human NI-308.5J10 antibody against C9orf72 dipeptide repeat protein peptide (GA)₁₅(■)(SEQ ID NO：66)、(GP)₁₅(▲)(SEQ ID NO：67)、(GR)₁₅ (SEQ ID NO：68)、(PA)₁₅(◆)(SEQ ID NO：69)、(PR)₁₅(●) (SEQ ID NO: 70) and BSA control (. DELTA.) EC₅₀Determined by indirect ELISA. Antibody NI-308.5J10 specifically recognizes C9orf72 DPR protein peptide (GA) with a binding affinity of 0.26nM₁₅(SEQ ID NO：66)。

FIG. 3: EC of BSA-coupled and unconjugated C9orf72 dipeptide repeat protein peptides₅₀And (4) measuring. Coupling (. tangle-solidup.) and uncoupling (■) of the C9orf72 dipeptide repeat protein peptide (GA) to BSA using an indirect ELISA humanized NI-308.5J10 antibody₁₅(SEQ ID NO：66)、(GP)₁₅(SEQ ID NO：67)、(GR)₁₅(SEQ ID NO：68)、(PR)₁₅(SEQ ID NO：69)、(PA)₁₅(SEQ ID NO: 70) or the half maximal Effective Concentration (EC) of the BSA control (. DELTA.)₅₀) The measurement of (1). Antibody NI-308.5J10 recognized BSA-conjugated (0.16nM) and unconjugated (0.23nM) C9orf72 DPR protein peptides with similar binding affinities.

FIG. 4: binding specificity analysis of human anti- (poly-GA) DPR antibody NI-308.5J10 to unrelated aggregated proteins. Determination of antibody binding to C9orf72(GA) by indirect ELISA₁₅(SEQ ID NO：66)、(GP)₁₅(SEQ ID NO：67)、(GR)₁₅(SEQ ID NO：68)、(PR)₁₅(SEQ ID NO: 70) and (PA)₁₅(SEQ ID NO: 69) binding of the peptide to 5 unrelated amyloidogenic proteins. Antibody NI-308.5J 10-shown to be related to C9orf72(GA)₁₅(SEQ ID NO: 66) without significant off-target binding to unrelated analytes. The NI-308.5J10 antibody was tested at a concentration of 20 nM.

FIG. 5: binding selectivity of NI-308.5J10 antibody to C9orf72 dipeptide repeat protein. Western blot analysis to determine the C9orf72 dipeptide repeat protein peptide (GA) conjugated to BSA by the human antibody NI-308.5J 10C 9orf72₁₅(SEQ ID NO：66)、(GP)₁₅(SEQ ID NO：67)、(GR)₁₅(SEQ ID NO：68)、(PA)₁₅(SEQ ID NO: 69) and (PR)₁₅(SEQ ID NO: 70). The C9orf72 poly-GA DPR protein was recognized by antibody NI-308.5J 10.

FIG. 6: antibody NI-308.5J 10C 9orf72 DPR protein repeat sequence length dependent binding. Determination of the C9orf72 poly-GA dipeptide repeat sequence of the human NI-308.5J10 antibody by indirect ELISADipeptide repeat length-dependent binding specificity and half-maximal effective concentration EC of protein peptides₅₀). Antibody NI-308.5J10 with EC at 13.8nM, 0.30nM and 0.29nM, respectively₅₀Binding affinity targeting DPR protein peptides (GA)₆(SEQ ID NO：80)、(GA)₁₀(SEQ ID NO: 79) and (GA)₂₀(SEQ ID NO: 82). Fig. 6 discloses SEQ ID NOs: 77. 76, 75, 74, 73, 72 and 71.

FIG. 7: binding of NI-308.5J10 to poly-GA C9orf72 dipeptide repeat protein peptide was characterized by biolayer interferometry. Measurement of protein peptide (GA) of C9orf72 dipeptide repeat sequence by antibody NI-308.5J10 using biolayer interferometry₁₅(SEQ ID NO: 66) binding constants KD, K _aAnd K_d. Shows NI-308.5J10 with fixed Synthesis (GA)₁₅Biolayer interferometry (BLI) sensorgram of binding of peptide (SEQ ID NO: 66). Antibodies were run at various concentrations: 30. 15, 7.5, 3.75 and 1.875 nM. Measurements were performed in triplicate. The sensorgram shows a single measurement of antibody concentration for each test, but the highest concentration additionally shows a second data set. Antibody NI-308.5J10 showed KD of (1.5. + -. 0.2) x 10^-10M，K_aIs (1.63 +/-0.05) x 10⁵M^-1s^-1And K_dIs (2.4 +/-0.4) x 10^-5s^-1。

FIG. 8: competitive binding of NI-308.5J10 and a reference human anti-poly-GA DPR antibody (NI-mAb reference) to poly-GA C9orf72 DPR peptide was characterized by biolayer interferometry. Determination of antibodies NI-308.5J10 and NI-mAb reference to C9orf72 DPR peptide (GA) Using biolayer interferometry₁₅(SEQ ID NO: 66). Biolayer interferometry (BLI) sensorgrams showing NI-308.5J10(A) and NI-mAb reference (B) against fixed synthesis (GA)₁₅Competitive binding of the peptide (SEQ ID NO: 66).

FIG. 9: integrity analysis of antibody NI-308.5J 10. SDS-PAGE analysis followed by Coomassie blue staining of 2 or 10 μ g of recombinant humanized NI-308.5J10 anti-C9 orf72 poly-GA DPR antibody. Two major bands corresponding to antibody heavy and light chains of expected size were detected.

FIG. 10: NI-308.5J10 detected pathological C9orf72 dipeptide repeat protein aggregates in FTLD patients. (A) Human NI-308.5J10 antibody revealed pathological neuronal cytoplasmic inclusions, neuronal nuclear inclusions and dystrophic neurites in the cerebellar granule cell layer in selected cases of C9orf 72-FTLD. In contrast, the non-nervous system control cerebellum stained negatively for NI-308.5J 10. (B) Representative high magnification images of neuronal C9orf72 DPR content in the cerebellar granule cell layer of selected C9orf72-FTLD cases detected by antibody NI-308.5J 10.

FIG. 11: the crystal structure of the NI-308.5J10 antibodies of mutations N54S, N54T, G55S, G55T and N75D have been mapped therein. As can be derived from the crystal structure, the post-translational modifications are remote from the binding site of the antibody.

FIG. 12: integrity analysis of NI-308.5J10 antibody variants. The engineered NI-308.5J10 antibody variant consisting of a combination of N75D light chain and each heavy chain mutant can be produced as an intact human IgG1 and also as a Fab. The size and homogeneity of the purified proteins were analyzed by SDS-PAGE, above: human IgG 1: lane 1, NI-308.5J10 WT/WT; lane 2, variant WT/N75D of NI-308.5J 10; lane 3, variant N54S/N75D of NI-308.5J 10; lane 4, variant N54T/N75D of NI-308.5J 10; lane 5, variant G55S/N75D of NI-308.5J 10; lane 6, variant G55T/N75D of NI-308.5J 10; the following: his-labeled Fab: lane 1, WT-Fab-6His/WT NI-308.5J 10; lane 2, NI-308.5J10 variant WT-Fab-6 His/N75D; lane 3, NI-308.5J10 variant N54S-Fab-6 His/N75D; lane 4, NI-308.5J10 variant N54T-Fab-6 His/N75D; lane 5, NI-308.5J10 variant G55S-Fab-6 His/N75D; lane 6, NI-308.5J10 variant G55T-Fab-6 His/N75D. All proteins showed the expected size with no significant aggregates or protein hydrolysates.

FIG. 13A: binding of DPR Ab-1 Fab to GA (e.g., poly (GA). As shown by surface plasmon resonance, Fab fragments of DPR Ab-1 bind (GA)₈The repeat sequence (SEQ ID NO: 81).

FIG. 13B: as shown by the crystal structure, the Fab fragment of DPR Ab-1 binds to the GA repeat peptide.

Detailed Description

The present invention relates generally to immunotherapy and non-invasive methods for detecting diseases and disorders associated with dipeptide repeat sequence (DPR) proteins and in particular their aggregated forms. More specifically, the present invention relates to recombinant human-derived monoclonal antibodies and DPR-binding fragments thereof, which are generated based on sequence information obtained from a selected human donor population and are capable of binding to such DPRs, in particular poly-glycine-alanine (Gly-Ala; GA) -DPR and proteins containing such DPR. The recombinant human monoclonal antibodies of the invention, as well as their synthetic and biotechnological derivatives, are advantageously characterized by specific binding to the altered C9orf72 with amplified hexanucleotide repeats forming the C9orf 72-dipeptide repeat (DPR). As shown in the examples, the recombinant antibodies of the invention are highly specific as diagnostic reagents for detecting DPR and/or pathological C9orf72 without generating false positives and due to the human origin of the sequences encoding at least the variable region and the CDRs, respectively, and it is reasonable to expect that maturation of the original antibody in humans is an effective and safe therapeutic agent.

I. Definition of

Unless otherwise indicated, terms as used herein are defined as provided in Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised version 2000 and reissue 2003, ISBN 0198506732. Furthermore, unless otherwise indicated, terms are as provided in WO 2016/050822 a2, and in particular definitions of the terms and expressions used herein are given in the section "i. definitions" on pages 26 to 53 (table 1 including CDR definitions on pages 39 and 40) to characterize the present invention, the disclosure of which is expressly incorporated herein by reference. The same applies to the general embodiments disclosed in WO 2016/050822 a2 for antibodies, polynucleotides and the like.

It should be noted that the term "an" entity refers to one or more of the entities; for example, "an antibody" is understood to mean one or more antibodies. Thus, the terms "a" or "an", "one or more" and "at least one" are used interchangeably herein.

The term "DPR", i.e. "dipeptide repeat" protein, is used herein to refer specifically to a repeat unit of two amino acids, particularly due to an amplified hexanucleotide repeat sequence in a gene, if not otherwise specified. The terms "DPR" and "DPR" are also used to refer collectively to all types and forms of DPR, such as GA, GR, GP, PA, PR, etc. Hereinafter, the present invention will be described mainly with respect to antibodies specifically recognizing DPRs comprising or consisting of: GA, e.g. (GA) n (where n is 1, 2, 3, 4, 5, 6 or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more), e.g. where n is between 6 and 15, including 6 and 15, e.g. where n is 15), e.g. having 15 repeats (GA) ₁₅) (SEQ ID NO: 66) GP, e.g. with 15 repetitive sequences (GP)₁₅) (SEQ ID NO: 67) GR, e.g. with 15 repetitive sequences (GR)₁₅) (SEQ ID NO: 68) or PR, e.g. with 15 repeating sequences (PR)₁₅) (SEQ ID NO: 70) or PA, e.g. having 15 repetitive sequences (PA)₁₅) (SEQ ID NO: 69) usually observed in the C9ORF72-DPR protein found in brain tissue of patients with FLTD or ALS. In embodiments, an anti-DPR antibody described herein specifically binds to a DPR comprising a GA repeat sequence. Although anti-C9 ORF72-DPR antibodies represent preferred embodiments, the invention generally provides anti-DPR protein antibodies and corresponding embodiments. Therefore, it is emphasized that in principle any embodiment and corresponding features disclosed herein and illustrated in the examples and figures are also meant to apply generally to any anti-DPR protein antibody, unless specifically applicable only to the anti-C9 ORF 72-DPR.

Another example of a DPR-associated disease is spinocerebellar ataxia 36, a subtype of slowly progressive neurodegenerative disorder and autosomal dominant cerebellar ataxia 1 (ADCA 1), characterized by adult-onset gait and limb ataxia, lower limb spasm, dysarthria, muscle tethering, tongue atrophy and hyperreflexia. Some affected individuals may also develop hearing loss; see, for example, Garcia-Murias et al Brain 135(2012), 1423-. The results indicate that spinocerebellar ataxia type 36 results from heterozygous amplification of the intron GGCCTG hexanucleotide repeat in the NOP56 gene on chromosome 20p 13; see, for example, Garcia-Murias et al Brain 135(2012), 1423-. Ikeda et al, Neurology 79(2012), 333-; kobayashi et al, am.J.hum.Gene.89 (2011), 121-130.

The term "C9 ORF 72" refers to an altered form of chromosome 9 open reading frame 72(C9ORF72) if not otherwise specifically indicated. The term "C9 ORF 72" is also commonly used to identify C9ORF72 hexanucleotide amplifications that result in a C9ORF 72-dipeptide repeat (DPR). Thus, the term is also used to refer to C9ORF 72-DPR. The term "C9 ORF 72" is also used to refer collectively to all types and forms of C9ORF72, such as mutated C9ORF 72. The letters added before the term C9ORF72 are used to indicate the organism from which a particular ortholog is derived, e.g. hC9ORF72 for human C9ORF72 or mC9ORF72 for murine origin.

The anti-DPR antibodies disclosed herein optionally bind C9ORF 72-dipeptide repeats (DPRs) and epitopes thereof. For example, disclosed herein are antibodies that specifically bind to a pathologically altered species of C9ORF72 or fragments thereof (i.e., dipeptide repeats that are non-regularly translated from the C9ORF72 transcript of the amplified intron C9ORF72 hexanucleotide repeat as well as aggregated forms of C9ORF72-DPR or fragments thereof). The term (pathologically) aggregated/aggregated of C9ORF72-DPR is used herein to refer specifically to the above forms. The term (pathological) "aggregated form" or "aggregate" as used herein describes the accumulated or cluster-formed product due to C9ORF72 error/pathological translation of the C9ORF72 transcript from the amplified intronic C9ORF72 hexanucleotide repeat. These aggregate, cumulative or cluster forms may be, consist essentially of, or consist of the C9ORF72-DPR protein and/or fragments thereof. As used herein, reference to an antibody that "specifically binds," "selectively binds," or "preferentially binds" C9ORF72-DPR refers to an antibody that does not bind to other unrelated proteins. The antibodies of the invention do not substantially recognize an unrelated amyloidogenic protein selected from the group consisting of: paired Helical Filament (PHF) -TAU, reciprocal response DNA binding protein 43(TDP-43), transthyretin (TTR), full-length amyloid precursor protein (flAPP), and/or Huntingtin (HTT). In one example, the C9ORF72-DPR antibodies disclosed herein can bind to DPR and/or C9ORF72-DPR or epitopes thereof, and show no binding above about 2-fold background of other proteins. An antibody that "specifically binds" or "selectively binds" to a variant of the DPR and/or C9ORF72-DPR protein refers to an antibody that does not bind to all variants of the C9ORF72-DPR protein, i.e. does not bind to at least one other C9ORF72 conformer. For example, disclosed herein are antibodies of a form that can preferentially bind to C9ORF72, which C9ORF72 displays a complimentary hexanucleotide repeat that forms a DPR in vitro or in tissues obtained from patients having or at risk of developing a disease associated with C9ORF 72.

The term "peptide" is understood to include within its meaning the terms "polypeptide" and "protein" (which are sometimes used interchangeably herein). Similarly, fragments of proteins and polypeptides are also contemplated and may be referred to herein as "peptides". However, the term "peptide" optionally denotes an amino acid polymer comprising at least 5 consecutive amino acids, such as at least 10 consecutive amino acids, such as at least 15 consecutive amino acids, such as at least 20 consecutive amino acids, such as at least 25 consecutive amino acids. In addition, the peptides according to the invention typically have no more than 100 consecutive amino acids, e.g., less than 80 consecutive amino acids or less than 50 consecutive amino acids.

The term "polypeptide" as used herein is intended to encompass both the singular "polypeptide" and the plural "polypeptide" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to one or more chains of two or more amino acids, are included in the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with, any of these terms.

The term "polypeptide" is also intended to refer to the product of a modification of a polypeptide following expression, including without limitation glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The polypeptide may be derived from a natural biological source or produced by recombinant techniques, but need not be translated from a specified nucleic acid sequence. It may be produced in any manner, including by chemical synthesis.

The polypeptides of the invention may have a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, but they do not necessarily have such a structure. Polypeptides having a defined three-dimensional structure are referred to as folded, and polypeptides that do not have a defined three-dimensional structure and can adopt many different conformations are referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-or nitrogen-containing side chain of an amino acid residue (e.g., a serine residue or an asparagine residue).

An "isolated" polypeptide or fragment, variant or derivative thereof is intended to be a polypeptide that is not in its natural surrounding environment. No particular level of purification is required. For example, an isolated polypeptide may be removed from its natural or native environment. For the purposes of the present invention, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated, as are native or recombinant polypeptides that have been isolated, fractionated or partially or substantially purified by any suitable technique.

"recombinant peptide, polypeptide or protein" refers to a peptide, polypeptide or protein produced by recombinant DNA techniques, i.e., produced by a cell, microorganism or mammal, transformed with an exogenous recombinant DNA expression construct encoding a fusion protein comprising the desired peptide. Proteins or peptides expressed in most bacterial cultures are generally glycan-free. The protein or polypeptide expressed in yeast may have a glycosylation pattern that is different from that of the protein or polypeptide expressed in mammalian cells.

As polypeptides of the invention, fragments, derivatives, analogs or variants of the above polypeptides are included as well as synthetic or biological variants and any combination thereof. The terms "fragment," "variant," "derivative," and "analog" include peptides and polypeptides having an amino acid sequence sufficiently similar to that of a native peptide. The term "sufficiently similar" means that a first amino acid sequence contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common domain and/or common functional activity. For example, amino acid sequences that comprise a common domain that is 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical are defined to be sufficiently similar. Optionally, the variant will be sufficiently similar to the amino acid sequence of a preferred peptide of the invention, in particular to an altered C9ORF72 protein, such as pathological C9ORF72-DPR as well as individual DPR proteins, variants, derivatives or analogs of any of them. Such variants typically retain the functional activity of the targeting construct of the invention. Variants include peptides that differ in amino acid sequence from the native and wt peptides, respectively, by one or more amino acid deletions, additions and/or substitutions. These may be naturally occurring variants as well as artificially designed variants.

Furthermore, when referring to an antibody or antibody polypeptide of the invention, the terms "fragment", "variant", "derivative" and "analogue" include any polypeptide that retains at least some of the antigen binding properties of the corresponding native binding molecule, antibody or polypeptide. Fragments of the polypeptides of the invention include, for example, proteolytic fragments as well as deletion fragments, in addition to the specific antibody fragments discussed elsewhere herein. The antibodies and variants of the antibodies of the invention include fragments as described above, and also include polypeptides having altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants may occur naturally or non-naturally. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides may include conservative or non-conservative amino acid substitutions, deletions, or additions. Derivatives of DPR protein-specific binding molecules (e.g., antibodies and antibody polypeptides of the invention) are polypeptides that have been altered to exhibit additional characteristics not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs". As used herein, a binding molecule or fragment thereof, an "derivative" of an antibody or antibody polypeptide refers to the subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. "derivatives" also include those peptides that contain one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxy lysine can be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine can be substituted for serine; and ornithine may be substituted for lysine.

Determining the similarity and/or identity of molecules:

the "similarity" between two peptides is determined by comparing the amino acid sequence of one peptide with the sequence of a second peptide. If the amino acid of one peptide is identical or a conservative amino acid substitution, it is similar to the corresponding amino acid of the second peptide. Conservative substitutions include those described in Dayhoff, M.O., eds, The Atlas of Protein sequences and Structure 5, National biological Research Foundation, Washington, D.C. (1978) and Argos, EMBO J.8(1989), 779-. For example, an amino acid belonging to one of the following groups represents a conservative change or substitution: -Ala, Pro, Gly, gin, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and-Asp, Glu.

"similarity" between two polynucleotides is determined by comparing the nucleic acid sequence of one polynucleotide to the sequence of the polynucleotide. If the nucleic acids of one polynucleotide are identical or if the nucleic acids are part of a coding sequence, the nucleic acids of the polynucleotide are similar to the corresponding nucleic acids of a second polynucleotide, comprising corresponding triplets of the nucleic acids encoding the same amino acid or encoding conservative amino acid substitutions. Using Karlin and Altschul (1993) proc.natl.acad.sci USA 90: 5873 the mathematical algorithm of 5877 optionally determines the percent identity or similarity between two sequences. This algorithm is incorporated into Altschul et al (1990) j.mol.biol.215: 403-.

The determination of percent identity or similarity is performed according to the standard parameters of the BLASTn program for BLAST polynucleotide searches and the BLASTp program for BLAST protein searches, as recommended for sequences of specific length and composition in the NCBI web pages and the "BLAST program selection guide".

BLAST polynucleotide searches were performed using the BLASTn program.

For the conventional parameters, the "maximum target sequence" box may be set to 100, the "short query" box may be checked, the "expectation threshold" box may be set to 1000, and the "word length" box may be set to 7 as recommended for short sequences (less than 20 bases) on NCBI web pages. For longer sequences, the "desired threshold" box may be set to 10, and the "word length" box may be set to 11. For the scoring parameter, the "match/no match score" can be set to 1, -2, and the "room cost" box can be set to linear. For filter and masking parameters, the "low complexity region" box may not be checked, the "species-specific repeat sequence" box may not be checked, the "mask for lookup table only" box may be checked, the "DUST filter settings" may be checked, and the "mask lower case" box may not be checked. Typically, "search for short nearly exact matches" may be used in this regard, which provides most of the above-described settings. Further information in this regard can be found in the BLAST program selection guide published on NCBI's web pages.

BLAST protein searches were performed using the BLASTp program. For the conventional parameters, the "maximum target sequence" box may be set to 100, the "short query" box may be checked, the "desired threshold" box may be set to 10, and the "word length" box may be set to "3". For the scoring parameter, the "matrix" box may be set to "BLOSUM 62", and the "room cost" box may be set to "Presence: 11, extension: 1 and the "composition adjustment" box may be set to "conditional composition score matrix adjustment". For the filter and masking parameters, the "low complexity region" box may not be checked, the "mask only for lookup tables" box may not be checked, and the "mask lower case" box may not be checked.

Both programs are modified, for example, in terms of the length of the sequences searched, according to the recommendations in the "BLAST program selection guide" published in HTML and PDF versions on NCBI web pages.

A polynucleotide:

the term "polynucleotide" is intended to encompass a single nucleic acid as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, such as messenger rna (mrna) or plasmid dna (pdna). Polynucleotides may comprise conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, as found in Peptide Nucleic Acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid fragments, such as DNA or RNA fragments, present in a polynucleotide. An "isolated" nucleic acid or polynucleotide means a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, for the purposes of the present invention, a recombinant polynucleotide encoding an antibody contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or (partially or substantially) purified polynucleotides in solution. An isolated RNA molecule includes an in vivo or in vitro RNA transcript of a polynucleotide of the invention. Isolated polynucleotides or nucleic acids according to the invention also include synthetically produced such molecules. In addition, the polynucleotide or nucleic acid may be or may include regulatory elements such as a promoter, ribosome binding site or transcription terminator.

As used herein, a "coding region" is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it may be considered part of the coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The two or more coding regions of the invention may be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. In addition, any vector may contain a single coding region, or may include two or more coding regions, e.g., a single vector may individually encode one immunoglobulin heavy chain variable region and one immunoglobulin light chain variable region. In addition, the vectors, polynucleotides or nucleic acids of the invention may encode heterologous coding regions, fused or unfused to nucleic acids encoding binding molecules, antibodies or fragments, variants or derivatives thereof. Heterologous coding regions include, but are not limited to, particular elements or motifs, such as secretion signal peptides or heterologous functional domains.

In some embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide may typically include a promoter and/or other transcriptional or translational control elements operably associated with one or more coding regions. Operably linked is that a coding region for a gene product (e.g., a polypeptide) is linked to one or more regulatory sequences in such a way that expression of the gene product is under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) "are operably associated" or "operably linked" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or with the ability of the DNA template to be transcribed. Thus, a promoter region will be operably associated with a nucleic acid encoding a polypeptide, so long as the promoter is capable of effecting transcription of the nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells. In addition to promoters, other transcriptional control elements such as enhancers, operators, repressors, and transcriptional termination signals may be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein.

Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to: transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter, in combination with intron-a), simian virus 40 (early promoter), and retroviruses, such as Rous sarcoma virus (Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit beta globulin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

Similarly, a variety of translational control elements are known to those of ordinary skill in the art. These translational control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly the inner ribosome entry site, or IRES, also known as CITE sequences).

In other embodiments, the polynucleotide of the invention is RNA, e.g., in the form of messenger RNA (mrna).

The polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions that encode secretion or signal peptides that direct the secretion of the polypeptide encoded by the polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the growing protein chain is initiated across the rough endoplasmic reticulum export. One of ordinary skill in the art will recognize that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the complete or "full-length" polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal sequence, such as an immunoglobulin heavy or light chain signal peptide, is used, or a functional derivative of such a sequence is used, which retains the ability to direct secretion of a polypeptide with which it is operably associated. Alternatively, a heterologous mammalian signal peptide, or functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted for the human Tissue Plasminogen Activator (TPA) or mouse-glucuronidase leader sequence.

"binding molecules" as used in the context of the present invention mainly relates to antibodies and fragments thereof, but may also refer to other non-antibody molecules binding to dipeptide repeat (DPR) proteins, which optionally bind to altered C9ORF72, in particular (pathologically) altered C9ORF72-DPR, including but not limited to hormones, receptors, ligands, Major Histocompatibility Complex (MHC) molecules, chaperones such as Heat Shock Proteins (HSP), and intercellular adhesion molecules such as cadherins, integrins, C-type lectins, and members of the immunoglobulin (Ig) superfamily. Thus, for the sake of clarity only and without limiting the scope of the invention, most of the following embodiments are discussed for antibodies and antibody-like molecules that represent preferred binding molecules for the development of therapeutic and diagnostic agents.

Antibody:

the terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody or immunoglobulin is a binding molecule comprising at least the variable domain of a heavy chain, and typically at least the variable domains of a heavy chain and a light chain. The basic immunoglobulin structure in vertebrate systems is relatively well understood; see, e.g., Harlow et al, Antibodies: a Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd edition 1988).

As discussed in more detail below, the term "immunoglobulin" encompasses a wide variety of different classes of polypeptides that can be biochemically distinguished. It will be understood by those skilled in the art that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (gamma, mu, alpha, delta, epsilon), with some subclasses (e.g., gamma 1-gamma 4) among them. The nature of this chain determines the "class" of antibodies as IgG, IgM, IgA IgG or IgE, respectively. The immunoglobulin subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known to confer functional specificity. In view of this disclosure, the skilled artisan will readily recognize the modified versions of each of these classes and isoforms, and thus are within the scope of the invention. All immunoglobulin classes are clearly within the scope of the present invention, and the following discussion will generally refer to immunoglobulin molecules of the IgG class. With respect to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides having a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides having a molecular weight of 53,000 to 70,000. These four chains are typically linked together in a "Y" configuration by disulfide bonds, with the light chain bracketing the heavy chain from the opening of the "Y" and all the way through the variable region.

Light chains are classified as either kappa or lambda (kappa, lambda). Each heavy chain class can be associated with a kappa or lambda light chain. Typically, the light and heavy chains are covalently bonded to each other, and when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the bifurcated end of the Y configuration to the C-terminus of the bottom of each chain.

Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this respect, it should be understood that light ((V)_L) Chain sum weight (V)_H) The variable domains of both chain portions determine antigen recognition and specificity. In contrast, the constant domains of the light Chain (CL) and heavy chains (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of constant region domains increases as they become further away from the antigen binding site or amino terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminal ends of the heavy and light chains, respectively.

As indicated above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on the antigen. I.e., V of the antibody_LDomains and V_HSubsets of domains or Complementarity Determining Regions (CDRs) are combined to form defined three-dimensional antibodiesA variable region of the primary binding site. This tetrabasic antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site consists of V_HAnd V_LThree CDRs on each of the chains. Any antibody or immunoglobulin fragment containing a structure sufficient to specifically bind to a DPR, particularly to the altered C9ORF72 that forms a C9ORF72-DPR, is interchangeably referred to herein as a "binding fragment" or an "immunospecific fragment.

In naturally occurring antibodies, the antibody comprises six hypervariable regions, sometimes referred to as "complementarity determining regions" or "CDRs," present in each antigen-binding domain, which are short, non-contiguous sequences of amino acids specifically positioned to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The "CDR" is flanked by four relatively conserved "framework" regions, or "FRs," which exhibit little intermolecular variability. The framework regions largely adopt a β -sheet conformation, and the CDRs form loops that connect to, and in some cases form part of, the β -sheet structure. Thus, the framework regions act to form a scaffold that provides for positioning the CDRs in the correct orientation through inter-chain, non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface complementary to an epitope on the immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody to its cognate epitope. The amino acids that make up the CDR and framework regions, respectively, can be readily identified by one of ordinary skill in the art for any given heavy or light chain variable region, as they have been precisely defined; see "Sequences of Proteins of Immunological Interest," Kabat, E., et al, U.S. department of health and public service, (1983); and Chothia and Lesk, J.mol.biol., 196(1987), 901-917, which are hereby incorporated by reference in their entirety.

In the case where there are two or more definitions of terms used and/or accepted in the art, the definition of terms as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining regions" ("CDRs") to describe non-contiguous antigen combining sites found in the variable regions of both heavy and light chain polypeptides. This particular region has been described by Kabat et al, U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by J.mol.biol., 196(1987), 901-917, which references are incorporated herein by reference, with the definitions including overlaps or subsets of amino acid residues when compared against each other. However, the use of either definition to refer to the CDRs of an antibody or variant thereof is intended to be within the scope of the terms as defined and used herein. Suitable amino acid residues encompassing the CDRs as defined by each of the above-cited references are listed as a comparison in table 1 below. The exact residue number encompassing a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular hypervariable region or CDR of the human IgG subtype of an antibody.

Table 1: CDR definition¹

	Kabat	Chothia
			VH CDR1	31-35	26-32
VH CDR2	50-65	52-58
			VH CDR3	95-102	95-102
VL CDR1	24-34	26-32
			VL CDR2	50-56	50-52
VL CDR3	89-97	91-96

¹The numbering defined for all CDRs in table 1 is according to the numbering convention recited by Kabat et al (see below).

Kabat et al also define a numbering system for the variable domain sequences applicable to any antibody. One of ordinary skill in the art can explicitly specify this "Kabat numbering" system for any variable domain sequence, without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al, the United states department of health and public service, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, reference to numbering specific amino acid residue positions in an antibody or antigen-binding fragment, variant, or derivative of the invention is according to the Kabat numbering system, however, this is theoretical and may not apply equally to each antibody of the invention. For example, depending on the position of the first CDR, subsequent CDRs may move in either direction.

Antibodies or fragments thereof (e.g., antigen-binding fragments or immunospecific fragments), variants or derivatives thereof of the invention include, but are not limited to: polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies, murinized or chimeric antibodies, single chain antibodies, epitope-binding fragments (e.g., Fab 'and F (ab') 2, Fd, Fv, single chain Fv (ScFv), single chain antibodies, disulfide-linked fvs (sdfv), fragments comprising VL or VH domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to the antibodies disclosed herein). ScFv molecules are known in the art and described, for example, in U.S. patent No. 5,892,019.

In one embodiment, the antibody of the invention is not an IgM or a derivative thereof having a pentavalent structure. In particular, in particular applications of the present invention, especially in therapeutic applications, IgM is less useful than IgG and other bivalent antibodies or corresponding binding molecules because it often shows non-specific cross-reactivity and very low affinity due to its pentavalent structure and lack of affinity maturation. In a particularly preferred embodiment, the antibody of the invention is not a polyclonal antibody, i.e. it consists essentially of one specific antibody species, rather than a mixture obtained from a plasma immunoglobulin sample.

Antibody fragments, including single chain antibodies, may comprise one or more variable regions, either alone or in combination with all or a portion of the following structures: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are DPR binding fragments comprising any combination of one or more variable regions and a hinge region, a CH1 domain, a CH2 domain, and a CH3 domain. The antibodies of the invention or immunospecific fragments thereof may be from any animal source, including birds and mammals. Optionally, the antibody is a human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibody. In another embodiment, the variable region may be derived from cartilaginous fish (e.g., from sharks).

As used herein, the term "heavy chain portion" or "heavy chain region" includes amino acid sequences derived from immunoglobulin heavy chains. The polypeptide comprising a heavy chain portion comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a binding polypeptide for use in the present invention may comprise: a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. Furthermore, a binding polypeptide used in the invention can lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As listed above, one of ordinary skill in the art will appreciate that these domains (e.g., heavy chain portions) can be modified to differ in their amino acid sequence from a naturally occurring immunoglobulin molecule.

In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof, disclosed herein, the heavy chain portions of one polypeptide chain of a multimer are identical to those heavy chain portions on a second polypeptide chain of the multimer. Alternatively, the heavy chain moiety-containing monomers of the invention are not identical. For example, each monomer may comprise a different target binding site, forming, for example, a bispecific antibody or diabody.

In another embodiment, the antibodies disclosed herein, or antigen-binding fragments, variants, or derivatives thereof, consist of a single polypeptide chain, such as an scFv, and will be expressed intracellularly (intrabody) for potential in vivo therapeutic and diagnostic applications.

The heavy chain portions of the binding polypeptides used in the diagnostic and therapeutic methods disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain portion of a polypeptide may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain portion may include a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain portion may include a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

As used herein, the term "light chain portion" or "light chain region" comprises amino acid sequences derived from an immunoglobulin light chain. Optionally, the light chain portion comprises at least one VL or CL domain.

The minimum size of a peptide or polypeptide epitope for an antibody is considered to be about four amino acids to five amino acids. The peptide or polypeptide epitope optionally contains at least seven, optionally at least nine, or optionally between at least about 15 to about 30 amino acids. Because a CDR can recognize an antigenic peptide or antigenic polypeptide in its tertiary form, the amino acids comprising the epitope need not be contiguous, and in some cases may not even be on the same peptide chain. In the present invention, the peptide or polypeptide epitope recognized by the antibody of the present disclosure contains DPR such as GA₁₅(SEQ ID NO: 66) of at least 4, at least 5, at least 6, at least 7, optionally at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 15 to about 30 contiguous or non-contiguous amino acids, as found in C9ORF 72-DPR. In other words, the antibody or biotechnological derivative thereof of the invention optionally recognizes a dipeptide consisting of two different amino acids X and X ' (XXX and XXX '; xaa ') with a number of repeats, e.g. 3 to 50, optionally 10 to 40, optionally 15 to 30 or optionally 15. Thus, an epitope or antigen recognized by an antibody of the invention or a biotechnological derivative thereof may be designated (XX') ₁₅。

"specifically binds" or "specifically recognizes" are used interchangeably herein and generally means that a binding molecule, e.g., an antibody, binds to an epitope via its antigen binding domain, and that binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to the epitope via its antigen binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to define the relative affinity by which an antibody binds to an epitope. For example, antibody "a" can be said to have a higher specificity for a given epitope than antibody "B", or antibody "a" can be said to bind to epitope "C" with a higher specificity than it does for the relevant epitope "D".

When present, the term "immunological binding properties" or other binding properties of an antibody to an antigen in all grammatical forms refers to the specificity, affinity, cross-reactivity and other binding properties of the antibody.

By "preferentially binds" is meant that a binding molecule, such as an antibody, specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or similar epitope. Thus, an antibody that "preferentially binds" a given epitope will be more likely to bind the given epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.

By way of non-limiting example, a binding molecule, e.g., an antibody, can be considered to preferentially bind a first epitope if it binds the first epitope with a dissociation constant (KD) that is less than the KD of the antibody to the second epitope. In another non-limiting example, an antibody can be considered to preferentially bind a first antigen if it binds the first epitope with an affinity that is at least an order of magnitude less than the KD of the antibody for the second epitope. In another non-limiting example, an antibody can be considered to preferentially bind a first epitope if it binds the first epitope with an affinity that is at least two orders of magnitude less than the KD of the antibody to the second epitope.

In another non-limiting example, an antibody may be considered to preferentially bind a first epitope if the binding molecule, e.g., an antibody, binds the first epitope with an off rate (k (off)) that is less than k (off) of the antibody directed against the second epitope. In another non-limiting example, an antibody may be considered to preferentially bind a first epitope if it binds the first epitope with an affinity that is at least one order of magnitude less than the k (off) of the antibody directed against the second epitope. In another non-limiting example, an antibody may be considered to preferentially bind a first epitope if it binds the first epitope with an affinity that is at least two orders of magnitude less than the k (off) of the antibody directed against the second epitope.

It can be said that the binding molecules disclosed herein, e.g. antibodies or antigen binding fragments, variants or derivatives are present at less than or equal to 5x 10^-2sec^-1、10^-2sec^-1、5x 10^-3sec^-1Or 10^-3sec^-1Binds to DPR or a fragment, variant or specific conformation thereof. Optionally, the antibodies of the invention can be said to be less than or equal to 5x 10^-4sec^-1、10^- ⁴sec^-1、5x 10^-5sec^-1Or 10^-5sec^-1、5x 10^-6sec^-1、10^-6sec^-1、5x 10^-7sec^-1Or 10^-7sec^-1Binds to the DPR protein or a fragment, variant or specific conformation thereof. In a particularly preferred embodiment, the DPR is a DPR related to C9ORF72, i.e.C 9ORF 72-DPR.

It can be said that the binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative disclosed herein, is greater than or equal to 10³M^-1sec^-1、5x 10³M^-1sec^-1、10⁴M^-1sec^-1Or 5x 10⁴M^-1sec^-1Binds to DPR or a fragment, variant or specific conformation thereof. Optionally, the antibody of the invention can be said to be greater than or equal to 10⁵M^-1sec^-1，5x10⁵M^-1sec^-1，10⁶M^-1sec^-1，or 5x 10⁶M^-1sec^-1or 10⁷M^-1sec^-1Binds to DPR or a fragment, variant or specific conformation thereof. In one embodiment, the binding molecule can be said to be greater than or equal to 10³M^-1sec^-1、5x 10³M^-1sec^-1、10⁴M^-1sec^-1Or 5x 10⁴M^-1sec^-1Binds to C9ORF72-DPR or a fragment, variant or specific conformation thereof. Optionally, the antibody of the invention can be said to be greater than or equal to 10⁵M^-1sec^-1、5x 10⁵M^-1sec^-1、10⁶M^-1sec^-1Or 5x 10⁶M^-1sec^-1Or 10⁷M^-1sec^-1Binds to C9ORF72-DPR or a fragment, variant or specific conformation thereof.

An antibody is said to competitively inhibit binding of a reference antibody to a given epitope if a binding molecule, e.g., an antibody, preferentially binds to the epitope to the extent that it blocks binding of the reference antibody to the epitope to some extent. Competitive inhibition can be determined by any method known in the art, for example, a competitive ELISA assay. An antibody can be said to competitively inhibit binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term "affinity" refers to a measure of the binding strength of an individual epitope to a CDR of a binding molecule (e.g., an immunoglobulin molecule). See, e.g., Harlow et al, Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2 nd edition (1988) pages 27-28. As used herein, the term "affinity" refers to the overall stability of the complex between immunoglobulin and antigen of a population, i.e., the functional combinatorial strength of a mixture of immunoglobulin and antigen; see, e.g., Harlow, pages 29-34. Affinity is related to the affinity of individual immunoglobulin molecules in a population with a specific epitope and also the valency of the immunoglobulin and antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repetitive epitope structure (e.g., a polymer) will be one of high affinity. The affinity or avidity (avidity) of an antibody for an antigen may be determined experimentally using any suitable method; see, e.g., Berzofsky et al, "Antibody-Antibody Interactions" In F unknown Immunology, Paul, W.E., eds., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W.H., Freeman and Company New York, N Y (1992), and methods described herein. Common techniques for measuring the affinity of an antibody for an antigen include ELISA, RIA and surface plasmon resonance. The measured affinity of a particular antibody-antigen interaction can vary if the measurement is performed under different conditions (e.g., salt concentration, pH). Thus, optionally, the affinity and other antigen binding parameters, e.g., KD, IC, are performed with standardized solutions of antibodies and antigens, as well as standardized buffers₅₀The measurement of (2).

Binding molecules of the invention, e.g., antibodies or antigen-binding fragments, variants or derivatives thereof, may also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody specific for one antigen to react with a second antigen; is a measure of the correlation between two different antigenic substances. Thus, an antibody is cross-reactive if it binds to an epitope other than the one that induced it to form. Cross-reactive epitopes usually contain many of the same complementary structural features as the inducing epitope and may in some cases actually fit better than the original.

For example, certain antibodies have a degree of cross-reactivity in which they bind related but non-identical epitopes, e.g., epitopes that are at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identical (as calculated using methods known in the art and described herein) to a reference epitope. An antibody may be said to have little or no cross-reactivity if it is unable to bind an epitope that is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identical to a reference epitope (as calculated using methods known in the art and described herein). An antibody may be considered "highly specific" for an epitope if it is unable to bind to any other analogue, ortholog or homologue of said epitope.

The binding molecules of the invention, e.g. antibodies or antigen-binding fragments, variants or derivatives thereof, may also be described or specified in terms of their binding affinity to DPRs and/or mutated C9ORF72 species and/or fragments thereof displaying C9ORF 72-DPRs. Preferred binding affinities include dissociation constants or Kd less than 5x 10 ^-2M、10^-2M、5x 10^-3M、10^-3M、5x 10^-4M、10^-4M、5x 10^-5M、10^-5M、5x 10^-6M、10^-6M、5x 10^-7M、10^-7M、5x 10^-8M、10^-8M、5x 10^-9M、10^-9M、5x 10^- ¹⁰M、10^-10M、5x 10^-11M、10^-11M、5x 10^-12M、10^-12M、5x 10^-13M、10^-13M、5x 10^-14M、10^-14M、5x 10^-15M or 10^-15M。

As previously indicated, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH 1 domain" includes the first (amino-most terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is the amino-terminal end of the hinge region of the immunoglobulin heavy chain molecule.

As used herein, the term "CH 2 domain" includes the portion of the heavy chain molecule that extends from about residue 244 to residue 360 of an antibody, for example, using conventional numbering schemes (residue 244 to residue 360, Kabat numbering system; and residue 231 to residue 340, EU numbering system; see Kabat EA, et al, cited above). The CH2 domain is unique in that it is not closely paired with another domain. While the two N-linked branched carbohydrate chains are between the two CH2 domains of the intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain with the CH2 domain. This hinge region comprises about 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains; see Roux et al J.Immunol.161(1998), 4083-.

As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group which can form a disulfide bond or a disulfide bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 region and the CL region are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system).

As used herein, the terms "linked," "fused," or "fused" are used interchangeably. These terms refer to the joining together of two or more elements or components by any means including chemical conjugation or recombinant means. By "in frame fusion" is meant the joining of two or more polynucleotide Open Reading Frames (ORFs) to form a continuous longer ORF in a manner that maintains the correct translational reading frame for the original ORF. Thus, a recombinant fusion protein is a single protein containing two or more segments corresponding to the polypeptide encoded by the original ORF (which segments are not normally so linked in nature) although the reading frames are thus rendered contiguous throughout the fusion segment, these segments may be physically or spatially separated, for example, by in-frame linker sequences. For example, polynucleotides encoding CDRs of an immunoglobulin variable region can be fused in frame, but isolated by polynucleotides encoding at least one immunoglobulin framework region or additional CDR regions, so long as the "fused" CDRs are co-translated as part of a contiguous polypeptide.

The term "expression" as used herein refers to the process by which a gene produces a biochemical, e.g., an RNA or a polypeptide. The process includes any manifestation of the functional presence of a gene in a cell, including but not limited to gene knock-outs and transient and stable expression. It includes, but is not limited to, transcription of genes into messenger RNA (mRNA), transfer RNA (trna), small hairpin RNA (shrna), small interfering RNA (sirna), or any other RNA product, and translation of mRNA into a polypeptide. If the final desired product is a biochemical, expression includes the production of the biochemical and any precursors. Expression of a gene results in a "gene product". As used herein, a gene product can be a nucleic acid, such as a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. The gene products described herein further include nucleic acids having post-transcriptional modifications, such as polyadenylation; or polypeptides with post-transcriptional modifications such as methylation, glycosylation, addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

As used herein, the term "sample" refers to any biological material obtained from a subject or patient. In one aspect, the sample may comprise blood, peritoneal fluid, CSF, saliva, or urine. In other aspects, the sample can include whole blood, plasma, serum, B cells enriched from a blood sample, and cultured cells (e.g., B cells from a subject). The sample may also comprise a biopsy or tissue sample comprising neural tissue. In still other aspects, the sample can comprise whole cells and/or lysates of cells. Blood samples can be collected by methods known in the art.

Diseases:

unless otherwise indicated, the terms "disorder" and "disease" are used interchangeably herein and include any undesirable physiological change in a subject, animal, isolated organ, tissue or cell/cell culture.

Frontotemporal lobar degeneration (FTLD) is a pathogenesis associated with atrophy of the frontal and temporal lobes of the brain. In addition, 50% of FTLD patients were also shown to have a positive family history and compared to Amyotrophic Lateral Sclerosis (ALS). As mentioned above, a common underlying cause of pathogenesis appears to be a heterozygously amplified hexanucleotide repeat located in C9ORF72 in FTLD and ALS patients. In particular, the resulting two amino acid repeat units (dipeptide repeats, DPR) are shown.

However, in several other diseases and/or disorders, amplified hexanucleotide repeats leading to repeats of two amino acids (DPRs) have also been reported. Such diseases include, but are not limited to, frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), FTLD-ALS, and/or spinocerebellar ataxia type 36 and associated symptoms therein.

In one embodiment of the invention, the antibody of the invention, the binding molecule having substantially the same binding specificity as any one thereof, the polynucleotide, vector or cell of the invention is used for the preparation of a medicament or diagnostic composition for the prophylactic and/or therapeutic treatment of a disease associated with DPR, for monitoring disease progression and/or therapeutic response, and for diagnosing a disease associated with DPR amyloidosis, including frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), FTLD-ALS, and/or spinocerebellar ataxia type 36.

In some embodiments, the antibodies of the invention bind to a pathological C9ORF 72-dipeptide repeat protein or an aggregated form thereof in FTLD patients. Thus, in one embodiment of the invention, the antibody, binding molecule, polynucleotide, vector or cell of the invention having substantially the same binding specificity as any one thereof is used in the manufacture of a medicament or diagnostic composition for prophylactic and/or therapeutic treatment of a disease associated with C9ORF72-DPR, for monitoring disease progression and/or therapeutic response, and for diagnosing a disease associated with C9ORF72-DPR or aggregated forms thereof, including frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS) and/or FTLD-ALS, and symptoms associated therewith.

Treatment:

as used herein, the terms "treatment" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the development of an undesirable physiological change or disorder, such as a cardiac defect. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "treatment" may also mean prolonging survival compared to that expected in the absence of treatment. Those in need of treatment include those already with the condition or disorder as well as those predisposed to the condition or disorder or those whose manifestation is to be prevented.

Unless otherwise indicated, the terms "drug", "drug" or "drug" are used interchangeably herein and shall include, but are not limited to, all (a) articles, drugs and preparations for internal and external use, and any substance or mixture of substances intended for use in the diagnosis, cure, mitigation, treatment or prevention of human or other animal diseases; and (B) articles, drugs and preparations (excluding food products) intended to affect the physical structure or any function of a human or other animal; and (C) an article intended to be a component of any of the articles specified in items (A) and (B). The terms "drug", "drug" or "drug" shall include the complete formulation of a formulation intended for use in a human or other animal, containing one or more "agents", "compounds", "substances" or "(chemical) compositions" and in other cases other pharmaceutically inactive excipients such as fillers, disintegrants, lubricants, glidants, binders, or ensuring that the "drug", "drug" or "drug" is readily transported, disintegrated, dissolved and bioavailable in the intended target site in the human or other animal, e.g., the skin, stomach or intestine. The terms "agent", "compound" or "substance" are used interchangeably herein and shall include, in a more specific context, but are not limited to, all pharmacologically active agents, i.e., agents that induce a desired biological or pharmacological effect or that are directed to the ability to be studied or tested for the ability to induce such a possible pharmacological effect by the methods of the present invention.

By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammalian subject, e.g., a human patient, in need of diagnosis, prognosis, prevention or treatment.

A drug carrier:

pharmaceutically acceptable carriers and routes of administration can be taken from the corresponding literature known to those skilled in the art. The pharmaceutical compositions of the present invention may be formulated according to methods well known in the art; preferably, see, e.g., Remington: the Science and Practice of Pharmacy (2000) by The University of Sciences in Philadelphia, ISBN 0-683-; banga, Therapeutic Peptides and Proteins: formulation, Processing, and Delivery systems, Taylor and Francis, 2 nd edition (2006), ISBN: 0-8493-1630-8. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions comprising such carriers may be formulated by well-known conventional methods. These pharmaceutical compositions may be administered to a subject at a suitable dose. The application of suitable compositions can be carried out in different ways. Examples include administration of compositions containing pharmaceutically acceptable carriers via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Aerosol formulations, such as nasal spray formulations, include purified aqueous or other solutions of the active agent with preservatives and isotonicity agents. Such formulations are optionally adjusted to a pH and isotonic state compatible with the nasal mucosa. Also contemplated in the present invention are pharmaceutical compositions for oral administration, such as single domain antibody molecules (e.g., "nanobodies) ^TM") and the like. These oral formulations may be in the form of tablets, capsules, powders, liquids or semi-solids. Tablets may contain solid carriers such as gelatin or adjuvants. Formulations for rectal or vaginal administration may be presented as suppositories with suitable carriers, see also O' Hagan et al, Nature Reviews, Drug Discovery 2(9) (2003), 727-735. Additional guidance regarding formulations suitable for a variety of application types can be found in Remington's Pharmaceutical Sciences, machine Publishing Company, philiadelphia, PA, 17 th edition (1985) and corresponding updates. For a brief review of drug delivery methods, see Langer, Science 249(1990), 1527-1533.

Antibodies of the invention

The present invention relates generally to anti-DPR, optionally anti-C9 orf72-DPR antibodies and DPR binding, i.e. DPR binding fragments, optionally displaying immunological binding properties and/or biological properties as outlined for the antibodies described herein, e.g. as illustrated in the examples, as well as biotechnological variants and derivatives thereof. In some embodiments, the anti-DPR antibody is a human or humanized antibody. In some embodiments, the anti-DPR antibody is a human monoclonal antibody. In some embodiments, the anti-DPR antibody is a human monoclonal antibody. According to the present invention, human monoclonal antibodies specific for poly-GA DPR have been cloned from a population of healthy human subjects. During the experiments performed according to the present invention, recombinant IgG antibodies derived from the original autoantibodies have been evaluated for their ability to bind to DPR and to other proteins, including Bovine Serum Albumin (BSA); see examples 3 to 9 and figures 2 to 8. As mentioned, in selected brain tissues of human C9orf72-FTLD patients, the subject antibodies may be shown to bind to DPR protein or aggregated forms thereof; see example 11 and figure 10. In addition, as shown in example 9 and FIG. 8, the subject antibodies were conjugated to aggregated C9orf72 poly-GA DPR (GA) ₁₅(SEQ ID NO: 66) binding was not blocked by previous binding of the reference anti-poly GA antibody (NI-mAb reference) to the target, thus pointing to the ability of the antibody to recognize conformational epitopes on poly-GA DPR aggregates, which are also accessible to other DPR protein and/or amyloidogenic protein co-aggregates.

Furthermore, anti-DPR antibodies, DPR binding fragments, synthetic or biotechnological derivatives or variants thereof may be optimized to have improved pharmacokinetic, manufacturability and stability properties. Thus, at least one amino acid in a CDR or variable region susceptible to modification selected from the group consisting of glycosylation, oxidation, deamination, peptide bond cleavage, isoaspartate formation and/or unpaired cysteines is substituted with a mutated amino acid which lacks such an alteration, or wherein at least one carbohydrate moiety is deleted or chemically or enzymatically added to the antibody, see e.g. Liu et al, j.pharm.sci.97(7) (2008), 2426-; beck et al, nat. rev. immunol.10(2010), 345-352; haberger et al, MAbs.6(2014), 327- & 339.

To investigate amino acid substitutions that may make the original antibody more stable and/or improve manufacturability while maintaining the basic binding properties of the parent subject antibody, poly- (GA) s have been prepared and analyzed ₈The crystal structure of the Fab fragment of the subject NI-308.5J10 antibody of peptide (SEQ ID NO: 81); see examples 12 to 16 and figures 11 and 12. To monitor the binding affinity of different variants of the original NI-308.5J10 antibody, Fab fragments were generated and tested to quantify the intrinsic monovalent affinity without the complexity from multivalent interactions. Since the modified Fab fragment substantially retains the affinity of the Fab fragment of the parent antibody, the corresponding intact IgG antibody is expected to be directed against poly- (GA)₁₅Parent antibodies to the peptide (SEQ ID NO: 66) have substantially the same binding affinity; see clause [14]And the accompanying examples.

Accordingly, the present invention relates generally to recombinant human-derived monoclonal anti-DPR antibodies and DPR-binding fragments thereof, synthetic and biotechnological derivatives and variants thereof, wherein the antibodies or DPR-binding fragments thereof comprise the following six Complementarity Determining Regions (CDRs) in their variable regions:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 (e.g., comprising SEQ ID NO: 78) or a variant thereof, wherein the variant comprises one or two amino acid substitutions,

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 4 or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(c) A VH-CDR3, said VH-CDR3 comprising SEQ ID NO: 5 or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(d) a VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8, or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(e) a VL-CDR2, said VL-CDR2 comprising SEQ ID NO: 9or a variant thereof, wherein said variant comprises one or two amino acid substitutions,

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10 or a variant thereof, wherein said variant comprises one or two amino acid substitutions;

wherein the antibody or DPR binding thereof exhibits any one of the properties set forth in the appended examples and figures for the subject NI-308.5J10 antibody and specific variants thereof, optionally wherein the antibody or DPR binding thereof has one or more of the properties as set forth in clause [1 []To [36 ]]The properties outlined in (1), optionally in combination, as indicated by the dependencies of the terms on each other. For example, in one embodiment, the antibody or DPR thereof binds to a polypeptide having at least 6 repeats (GA) as translated from the open reading frame 72(C9orf72) gene of chromosome 9 ₆A dipeptide repeat sequence (DPR) of polyglycine-alanine (GA) (SEQ ID NO: 80). A synthetic or biotechnological derivative or variant of the subject NI-308.5J10 can contain one, two, three, four, five, or six variant CDRs as described herein. For example, the synthetic or biotechnological derivative or variant antibody or DPR binding thereof may contain three, optionally two or optionally only one variant VH-CDR, while the VL-CDR remains unchanged or only one VL-CDR represents a variant, or vice versa. Additionally or alternatively, an antibody of the invention or a DPR-binding fragment thereof comprises in its variable region

(a) Comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof (V)_H) A chain, wherein the variant comprises one or more amino acid substitutions; and

(b) comprises the amino acid sequence of SEQ ID NO: 7 or a variant thereof (V)_L) A chain, wherein the variant comprises one or more amino acid substitutions; optionally wherein

The V is_HAnd VL chain amino acid sequences are identical to SEQ ID NO: 2 and 7 are at least 90% identical.

Preferred criteria for selecting appropriate amino acids for substitutions and positions in the CDRs are shown in figure 1 and explained in the legend above in figure 1.

As shown in examples 12 to 16, translation of the NI-308.5J10 hIgG1 antibody has been identifiedThe post-modification, i.e. light chain glycosylation and deamidation of heavy chain Asn54, has been removed by a corresponding amino substitution. Thus, in one embodiment of the antibody or DPR-binding fragment thereof of the invention, the CDRs do not contain deamidable asparagine (N) and/or glutamine (Q) and/or said V_HAnd/or V_LThe chain amino acid sequence does not contain occupied glycosylation sites. In one embodiment, said V is_HAnd/or V_LOne or more glycosylation sites in the chain amino acid sequence have been mutated to a glycosylation site that cannot be occupied.

In a preferred embodiment, the one, two or more amino acid substitutions in the antibody or DPR-binding fragment thereof are selected from the group consisting of

(a) Replacing asparagine (N) or glutamine (Q) that is susceptible to deamidation with a non-susceptible amino acid;

(b) replacing a small flexible amino acid directly adjacent to a readily deamidated N or Q with a larger amino acid, optionally wherein the adjacent amino acid is glycine (G);

(c) substituting at least one amino acid that results in removal of a glycosylation site, optionally wherein the at least one amino acid is within a glycosylation motif NXS or NXT; and/or

(d) (ii) substituting one or more amino acids that are conservative amino acid substitutions; optionally, wherein the amino acid substitutions of (a) and (b) are present in VH-CDR2 and the amino acid substitution of (c) is present in V_LIn the chain.

Here, furthermore, the amino acids for substitution are optionally selected according to the above considerations, with the aim of improving the stability and manufacturability of the subject antibodies and DPR-binding fragments thereof, respectively. Optionally, according to embodiments in VH-CDR2, a nucleic acid sequence corresponding to SEQ ID NO: 2 and/or an asparagine (N) at position 54 corresponding to SEQ ID NO: 2 with another amino acid, optionally wherein the asparagine (N) is substituted with serine (S) or threonine (T), and/or wherein the glycine (G) is substituted with serine (S) or threonine (T); and/or at said V_LIn the strand, the amino acid sequence corresponding to SEQ id no: 7 by another amino acid, optionally wherein the asparagine (N) at position 75 ofAsparagine (N) is substituted with aspartic acid (D).

Optionally, the anti-DPR antibody, DPR binding fragment or biotechnological derivative or variant thereof, if analyzed in IgG format, optionally IgG1 for binding to DPR protein (GA)₆(SEQ ID NO: 80) has an EC corresponding to ≦ 15nM ₅₀(half maximal effective concentration) value of binding affinity-see example 7-and/or for binding to DPR protein (GA)₁₀(SEQ ID NO：79)、(GA)₁₅(SEQ ID NO: 66) and/or (GA)₂₀(SEQ ID NO: 82) has an EC of ≦ 5nM, optionally ≦ 2nM, optionally ≦ 1nM or optionally ≦ 0.5nM₅₀Binding affinity of the value; see examples 3, 4 and 7 and figures 2 and 6. In one embodiment, the subject antibody binds to a poly-GA peptide only when the number of repeated sequences, n, is ≦ 6; see example 7 and figure 6. Additionally or alternatively, at least the antibody in IgG form binds to poly- (GA) with an affinity KD of about (0.5-2.0nM) as determined by biolayer interferometry₁₅(SEQ ID NO: 66), optionally a KD of about (0.05-0.5nM) and optionally a KD of about (0.1-0.2nM) binds to poly- (GA)₁₅Peptide (SEQ ID NO: 66) with an association rate constant of (K)_a＝0.5-5x 10⁵M^-1s^-1) And a dissociation constant of (K)_d＝1-5x 10^-5s^-1) (ii) a See example 8 and figure 7.

Thus, for high affinity and mentioned EC₅₀And a KD value, said anti-DPR antibody, DPR-binding fragment or biotechnological derivative or variant thereof optionally further comprising a CDR or V as described_HAnd V_LChain amino acid sequence optionally a heterologous polypeptide sequence, optionally wherein the polypeptide sequence comprises a human constant domain, optionally of the IgG type, optionally of the IgG1 class or isotype.

On the other hand, DPR binding fragments (especially Fab fragments) of the subject NI-308.5J10 antibody prove particularly useful for designing and studying synthetic and biotechnological derivatives or variants, optionally in the case of smaller poly GA repeats, i.e. (GA)₈(SEQ ID NO: 81); see examples 13 to 16 and figures 11 and 12. Therefore, in another implementationIn this protocol, the antibody or DPR binding fragment thereof of the invention is directed to poly- (GA), as determined by Surface Plasmon Resonance (SPR)₈(SEQ ID NO: 81) has a binding affinity corresponding to K_D(dissociation constant) less than 30nM, where K_a(association rate) less than 5x 10⁵M^- ¹s^-1And K is_d(dissociation rate) less than 10x 10^-3s^-1Optionally wherein the DPR binding fragment pair has a binding affinity corresponding to K as determined by Surface Plasmon Resonance (SPR)_D(dissociation constant) is 10nM to 30nM, where K_a(association rate) 1 to 5x 10⁵M^-1s^-1And K is_d(dissociation rate) 2.5 to 10x 10^-3s^-1. Additionally or alternatively, the antibody or DPR-binding fragment thereof is optionally characterized by a Fab fragment having a thermostability and melting temperature T, respectively, in the range of 78 ℃ -82 ℃, optionally in the range of about 79 ℃ -81 ℃, as determined by differential scanning calorimetry (VP-DSC) _m(ii) a See example 16.

Some antibodies are capable of binding to a variety of biomolecules, such as proteins. As the skilled person will appreciate, the term specificity is used herein to indicate that other biomolecules than DPR do not significantly bind to the antibodies of the invention. Optionally, the level of binding to biomolecules other than DPR results in a binding affinity that is at most only 20% or less, 10% or less, only 5% or less, only 2% or less, or only 1% or less (i.e., at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold lower, or any other value outside of this range) of the affinity for DPR. In particular, as mentioned above and illustrated in the examples and figures, according to the present invention, said anti-DPR antibody or DPR-binding fragment thereof or biotechnological derivative or variant thereof optionally exhibits 1, 2, 3, 4 or all 5 of the following binding properties: (i) identifying poly- (GA)₁₅Conformational epitopes on the peptide (SEQ ID NO: 66), i.e., capable of binding poly-GADPR aggregates after previous binding of different anti-poly-GA DPR antibodies (example 9 and FIG. 8); (ii) bind to poly-GA peptides coupled to BSA carrier protein with substantially the same affinity as the corresponding hydrophobically coated peptides (examples 3 and 4 and figures) 2 and 3); (iii) at least those tested in example 5 and shown in figure 4 have substantially no or minimal cross-reactivity with unrelated amyloidogenic proteins; (iv) aggregates comprising DPR-containing proteins translated from the C9orf72 gene in the granular cell layer of the cerebellum of C9orf72-FTLD patients can be bound (example 11 and fig. 10).

As mentioned previously, aggregation of DPR proteins in the frontal and temporal lobes of the brain is a hallmark of the neurodegenerative disease FTLD. Patients with neuronal cytoplasmic inclusions, neuronal nuclear inclusions, and DPR aggregates in dystrophic neurites in the cerebellar granule cell layer often exhibit altered cognitive function. In particular, as described above, patients with FTLD exhibit dementia, behavioral and personality, language dysfunction and/or psychiatric changes due to degeneration of the frontal and temporal cortex. Accordingly, in one embodiment, the antibodies of the invention, or DPR-binding fragments thereof, are useful for treating diseases and/or disorders associated with DPR. In a preferred embodiment, the antibodies of the invention or DPR-binding fragments thereof are useful for treating FTLD and symptoms thereof. The therapeutic use of the subject antibodies or DPR-binding fragments thereof of the invention can be validated in cellular assays (such as those described in the background section) (see also example 17), and optionally, such that if administered to a transgenic C9orf72 mouse model, the antibodies are capable of ameliorating at least one symptom of a pathological hallmark of C9orf72 disease, such as neuronal loss, behavioral abnormalities, dyskinesias, and decreased survival (example 18).

The corresponding nucleotide sequences encoding the variable regions identified above are listed in table 2 below. V_HAnd V_LAn exemplary set of CDRs of the amino acid sequences of the chains is shown in any of figures 1A-F to. Thus, the present invention provides a novel genus of anti-DPR antibodies, exemplified by antibodies or DPR-binding fragments thereof comprising in their variable regions

(i) The following six CDRs:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 (e.g., comprising SEQ ID NO: 78),

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof,

(d) a VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,

(e) a VL-CDR2, the VL-CDR2 comprising SEQ id no: 9, or a pharmaceutically acceptable salt thereof, wherein,

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10;

(ii) the following six CDRs:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 (e.g., comprising SEQ ID NO: 78),

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof, wherein,

(d) A VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,

(e) a VL-CDR2, the VL-CDR2 comprising SEQ id no: 9, or a pharmaceutically acceptable salt thereof, wherein,

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10;

(iii) the following six CDRs:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 (e.g., comprising SEQ ID NO: 78),

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 19, or a pharmaceutically acceptable salt thereof, wherein,

(d) a VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,

(e) a VL-CDR2, the VL-CDR2 comprising SEQ id no: 9, or a pharmaceutically acceptable salt thereof, wherein,

(f) a VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10; or

(iv) The following six CDRs:

(a) a VH-CDR1, said VH-CDR1 comprising SEQ ID NO: 3 (e.g., comprising SEQ ID NO: 78),

(b) a VH-CDR2, said VH-CDR2 comprising SEQ ID NO: 22, or a pharmaceutically acceptable salt thereof,

(d) a VL-CDR1, said VL-CDR1 comprising SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,

(e) a VL-CDR2, the VL-CDR2 comprising SEQ id no: 9, or a pharmaceutically acceptable salt thereof, wherein,

(f) A VL-CDR3, said VL-CDR3 comprising SEQ ID NO: 10;

optionally, wherein the antibody or DPR binding fragment comprises in its variable region

(i) As shown in SEQ ID NO: 2 and SEQ ID NO: v shown in 24_HAnd V_LA chain amino acid sequence;

(ii) as shown in SEQ ID NO: 12 and SEQ ID NO: v shown in 24_HAnd V_LA chain amino acid sequence;

(iii) as shown in SEQ ID NO: 15 and SEQ ID NO: v shown in 24_HAnd V_LA chain amino acid sequence;

(iv) as shown in SEQ ID NO: 18 and SEQ ID NO: v shown in 24_HAnd V_LA chain amino acid sequence;

(v) as shown in SEQ ID NO: 21 and SEQ ID NO: v shown in 24_HAnd V_LA chain amino acid sequence;

(vi) as shown in SEQ ID NO: 12 and SEQ ID NO: v shown in 7_HAnd V_LA chain amino acid sequence;

(vii) as shown in SEQ ID NO: 15 and SEQ ID NO: v shown in 7_HAnd V_LA chain amino acid sequence;

(viii) as shown in SEQ ID NO: 18 and SEQ ID NO: v shown in 7_HAnd V_LA chain amino acid sequence;

(ix) as shown in SEQ ID NO: 21 and SEQ ID NO: v shown in 7_HAnd V_LA chain amino acid sequence.

However, as already discussed above, the person skilled in the art is well aware of the fact that: additionally or alternatively, CDRs may be used which differ in their amino acid sequence from the amino acid sequence shown in any one of figures 1A-F, by one or two, or even more amino acids in the case of CDR2 and CDR 3. Thus, in one embodiment, antibodies of the invention, biotechnological derivatives and variant anti-DPR antibodies and DPR fragments thereof are provided, comprising in their variable regions CDRs as depicted in any one of figures 1A-F, wherein one or more, optionally not more than one or two of their CDRs comprise one or more, optionally not more than two amino acid substitutions; see also above.

As shown in the examples, one or two amino acid substitutions in VH-CDR2 did not affect the binding affinity and properties of the original antibody. With respect to further or other amino acid substitutions in the CDRs and in the variable heavy and light chain amino acid sequences, respectively, conservative amino acid substitutions are optionally made, e.g., according to the most frequently exchanged amino acids analyzed and described by Mirsky et al, mol.biol.evol.35(2014), 806-; see figure 6, page 813 of Mirsky et al. In this case, preliminary analysis of the CDRs of other human anti-poly-GA DPR antibodies with similar and different binding properties revealed that certain positions within the CDRs of the subject antibodies, as well as amino acid substitutions similar to those directed against VH-CDR2, unaffected the unique binding properties of the antibodies. The corresponding positions of preferred amino acid substitutions within the CDRs are shown in bold and italics in fig. 1, including those shown only in bold in VH-CDR 2.

In particular, within VH-CDR1, D may be substituted with S and/or S may be substituted with T; within VH-CDR3, V may be substituted with E, T may be substituted with S and/or M may be substituted with V; within VL-CDR1, R may be substituted with K, P may be substituted with S, R may be substituted with E, S may be substituted with G, and T may be substituted; within VL-CDR2, S may be substituted with A and/or A may be substituted with G; and in VL-CDR3, G may be substituted with a, L may be substituted with I, and P may be substituted with S, which may be substituted with P. As stated, optionally, amino acid substitutions belonging to the same category in one or optionally both of the models LG and AB shown in Mirsky et al (2014), supra, fig. 6, are selected, wherein the LG model is preferred for the trend towards preserving amino acid properties, and wherein the amino acid substitutions are optionally selected such that the physicochemical properties of the original amino acid, i.e. the hydrophobicity, polarity or charge properties, are substantially preserved or, for example, in case two or more amino acid substitutions are made, they compensate each other so as to together provide the physicochemical properties of the surface.

Provided herein is an anti-DPR antibody or fragment thereof, e.g., DPR Ab-1. The amino acid sequence information of DPR Ab-1 is shown in Table 12. In some embodiments, the anti-DPR antibody or antigen-binding fragment thereof binds to a DPR described herein, e.g., chromosome 9 open reading frame 72(C9orf72) dipeptide repeat (DPR) protein. In some embodiments, the DPR protein comprises a polyglycine-alanine (GA) repeat, e.g., a poly- (GA) n repeat, wherein n is 1, 2, 3, 4, 5, 6, or greater (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or greater). In some embodiments, the DPR protein comprises a poly- (GA) n repeat sequence, wherein n is between 6 and 15, including 6 and 15, for example wherein n is 15. In embodiments, the anti-DPR antibody or fragment thereof comprises DPR Ab-1 or a fragment thereof (e.g., an antigen-binding fragment). In some embodiments, the italicized asparagine residues in table 12 are glycosylated; in other embodiments, the italicized asparagine residues in table 12 are unglycosylated.

TABLE 12 amino acid sequence information of DPR Ab-1

Framework (FR) regions and Complementarity Determining Regions (CDRs) are shown in table 12, with the CDRs underlined. The Kabat numbering scheme is used (see http:// www.bioinf.org.uk/abs/; Kabat et al, U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983), cited in the mentioned network references and given in Table 1 at pages 39 and 40 of WO 2016/050822A 2, incorporated herein by reference unless otherwise specified, the reference to the numbering of specific amino acid residue positions in an antibody of the invention, or a DPR-binding fragment, variant or derivative thereof, is according to the Kabat numbering system, however, this is theoretical and may not apply equally to each antibody of the invention. I.e. the Variable Heavy (VH) and Variable Light (VL) chain amino acid sequences of the antibody DPRAb-1 at positions suitable for determining the correct CDR sequences according to Kabat, which applies to the definition of the claimed antibodies and DPR-binding fragments thereof.

Provided herein is a composition comprising an anti-DPR (e.g., anti-C9 ORF72 DPR) antibody or fragment thereof (e.g., that binds, e.g., specifically binds and/or binds with high affinity to a poly- (GA) n repeat, e.g., a poly- (GA) n repeat described herein), wherein the anti-DPR antibody or fragment thereof comprises:

(i) a heavy chain comprising SEQ ID NO: 37 or 38 (or an amino acid sequence which is at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37 or 38), consists of or essentially consists of,

optionally, wherein the heavy chain amino acid sequence (e.g., heavy chain mature sequence) is no more than 452 amino acids in length, and/or wherein the heavy chain does not comprise a lysine residue at the C-terminus of its amino acid sequence, and/or wherein the heavy chain has a glycine at the C-terminus of its amino acid sequence, and/or wherein the heavy chain comprises a heavy chain variable region amino acid sequence of no more than 123 amino acids in length;

(ii) a heavy chain comprising a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 39 (or an amino acid sequence which is at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39), consists of or consists essentially of,

(iii) a heavy chain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40 (or an amino acid sequence which is at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 40), consists or essentially consists thereof,

(iv) a heavy chain variable region amino acid sequence comprising SEQ ID NO: 40 (or an amino acid sequence which is at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 40), consists or essentially consists thereof,

Optionally, wherein the heavy chain variable region amino acid sequence is no more than 123 amino acids in length;

(v) a light chain comprising SEQ ID NO: 41 or 42 (or an amino acid sequence which is at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41 or 42), consists or essentially consists thereof,

optionally, wherein the light chain amino acid sequence (e.g., light chain mature sequence) is no more than 214 amino acids in length, and/or wherein the light chain comprises a light chain variable region amino acid sequence no more than 107 amino acids in length;

(vi) a light chain comprising a light chain constant domain comprising the amino acid sequence of SEQ ID NO: 43 (or an amino acid sequence at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 43), consists of or essentially consists of,

(vii) a light chain comprising a light chain variable region amino acid sequence comprising the amino acid sequence of SEQ ID NO: 44 (or an amino acid sequence at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 44), consists or consists essentially of,

Optionally, wherein the light chain amino acid sequence (e.g., light chain mature sequence) is no more than 214 amino acids in length, and/or wherein the light chain variable region amino acid sequence is no more than 107 amino acids in length;

(viii) a light chain variable region amino acid sequence comprising SEQ ID NO: 44 (or an amino acid sequence at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 44), consists or consists essentially of,

optionally, wherein the light chain variable region amino acid sequence is no more than 107 amino acids in length;

(ix) a heavy chain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 45); SEQ ID NO: 46 (or an amino acid sequence at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 46), and the CDR2 amino acid sequence of SEQ ID NO: 47 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47),

(x) A heavy chain variable region comprising SEQ ID NO: 45 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 45); SEQ ID NO: 46 (or an amino acid sequence at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 46), and the CDR2 amino acid sequence of SEQ ID NO: 47 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47),

optionally, wherein the heavy chain variable region amino acid sequence is no more than 123 amino acids in length;

(xi) A light chain comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48 (or an amino acid sequence at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 48); SEQ ID NO: 49 (or an amino acid sequence at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49), and the CDR2 amino acid sequence of SEQ ID NO: 50 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 50),

(xii) A light chain variable region comprising SEQ ID NO: 48 (or an amino acid sequence at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 48); SEQ ID NO: 49 (or an amino acid sequence at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49), and the CDR2 amino acid sequence of SEQ ID NO: 50 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 50),

optionally, wherein the light chain variable region amino acid sequence is no more than 107 amino acids in length; and/or

(xiii) A heavy chain and a light chain, the light chain comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48); SEQ ID NO: 49 (or an amino acid sequence at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49), and the CDR2 amino acid sequence of SEQ ID NO: 50 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 50),

Optionally, wherein the heavy chain amino acid sequence (e.g., heavy chain mature sequence) is no more than 452 amino acids in length, and/or wherein the heavy chain does not comprise a lysine residue at the C-terminus of its amino acid sequence, and/or wherein the heavy chain has a glycine at the C-terminus of its amino acid sequence, and/or wherein the heavy chain comprises a variable region amino acid sequence of no more than 123 amino acids in length; and/or optionally, wherein the light chain amino acid sequence is no more than 214 amino acids in length, and/or wherein the light chain variable region amino acid sequence is no more than 107 amino acids in length.

In one embodiment, provided herein is a polynucleotide, optionally linked to a heterologous nucleic acid, wherein (i) the polynucleotide encodes an immunoglobulin variable heavy chain having a VH-CDR as described herein, and wherein the immunoglobulin variable heavy chain is capable of (e.g., specifically and/or with high affinity) binding to a DPR (e.g., poly- (GA) n) as described herein when paired with an immunoglobulin variable light chain comprising an amino acid sequence as listed in table 12, and/or (ii) the polynucleotide encodes an immunoglobulin variable light chain having a VL-CDR as described herein, and wherein the immunoglobulin variable light chain is capable of (e.g., specifically and/or with high affinity) binding to a DPR (e.g., poly- (GA) n).

In some aspects, provided herein is an antibody or fragment thereof comprising a signal peptide at the N-terminus. In some embodiments, the signal peptide comprises amino acid sequence MGWSLILLFLVAVATRVLS (SEQ ID NO: 59) or a sequence identical to SEQ ID NO: 59, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical. In other embodiments, the signal peptide comprises the amino acid sequence MDMRVPAQLLGLLLLWFPGSRC (SEQ ID NO: 60) or a sequence identical to SEQ ID NO: 60, at least 95%, such as 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence.

In embodiments, the heavy chain thereof does not comprise a lysine residue at the C-terminus of its amino acid sequence. In embodiments, the heavy chain thereof has a glycine at the C-terminus of its amino acid sequence.

In embodiments, the heavy chain amino acid sequence (e.g., heavy chain mature sequence) is no more than 452 amino acids in length (e.g., 452 amino acids in length). In embodiments, the heavy chain does not comprise a lysine residue at the C-terminus of its amino acid sequence. In embodiments, the heavy chain has a glycine at the C-terminus of its amino acid sequence. In embodiments, the heavy chain variable region amino acid sequence is no more than 123 amino acids in length (e.g., 123 amino acids in length). In embodiments, the light chain variable region amino acid sequence is no more than 214 amino acids in length (e.g., 214 amino acids in length. in embodiments, the light chain variable region amino acid sequence is no more than 107 amino acids in length (e.g., 107 amino acids in length).

In some embodiments, the antibody or fragment thereof described herein comprises a heavy chain having an IgG1 isotype, e.g., a human IgG1(hIgG1) isotype. In embodiments, the heavy chain comprises allotype G1ml, 17.

In some embodiments, an antibody or fragment thereof described herein comprises a light chain having a kappa isotype, e.g., a human kappa isotype. In embodiments, the light chain comprises allotype Km 3.

In some embodiments, an antibody or fragment thereof described herein is linked to a drug; or detectably labeled with a label, such as an enzyme, radioisotope, fluorophore, tag, heavy metal, and/or a label.

The antibodies of the invention may be of human origin, in particular for therapeutic applications. Alternatively, the antibodies of the invention are rodent, rodent-humanized or chimeric rodent-human antibodies, optionally murine, murinized or chimeric murine-human antibodies or rat, murinized or chimeric murine-human antibodies, which are particularly useful for animal regime diagnostic methods and studies. In one embodiment, the antibody of the invention is a chimeric rodent-human or rodent-humanized antibody.

As discussed above, in addition to full antibodies, the antibodies of the invention may also exist in a variety of forms; including, for example, Fv, Fab and F (ab)2 and in single chain form; see, for example, international application WO 88/09344. Accordingly, in one embodiment, there is provided an antibody of the invention selected from the group consisting of: single-chain Fv fragment (scFv), F (ab ') fragment, F (ab) fragment and F (ab') ₂And (3) fragment.

The antibodies of the invention or their corresponding immunoglobulin chains may be further modified using conventional techniques known in the art, e.g., by using amino acid deletions, insertions, substitutions, additions and/or recombinations, alone or in combination, and/or any other modifications known in the art. Methods for introducing such modifications into DNA sequences that underlie the amino acid sequence of an immunoglobulin chain are well known to those skilled in the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Modifications to the antibodies of the invention include chemical and/or enzymatic derivatization at one or more of the constituent amino acids, including side chain modifications, backbone modifications, and N-and C-terminal modifications, including acetylation, hydroxylation, methylation, amidation, and attachment of carbohydrate or lipid moieties, cofactors, and the like. Likewise, the invention encompasses the generation of chimeric proteins comprising the described antibodies or some fragment thereof fused at the amino terminus to a heterologous molecule (such as an immunostimulatory ligand) at the carboxy terminus. See, for example, International application WO00/30680 for corresponding technical details.

As known to those of ordinary skill in the art, an antibody or DPR-binding fragment, synthetic or biotechnological variant or derivative thereof of the present invention may comprise a constant region that mediates one or more effector functions. For example, binding of the C1 component of complement to the constant region of an antibody activates the complement system. Activated complement is important for opsonization and lysis of cellular pathogens. Activation of complement also stimulates inflammatory responses and may also involve autoimmune hypersensitivity. In addition, antibodies bind to receptors on various cells via the Fc region, where the Fc receptor site on the Fc region of the antibody binds to an Fc receptor (FcR) on the cell. There are a number of Fc receptors that have specificity for different classes of antibodies, including IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). Binding of antibodies to Fc receptors on cell surfaces triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

Accordingly, certain embodiments of the invention include antibodies, or DPR-binding fragments, variants, or derivatives thereof, in which at least a portion of one or more constant region domains has been deleted or otherwise altered so as to provide a desired biochemical property, such as reduced effector function, ability to non-covalently dimerize, ability to localize at sites of aggregation and deposition of DPR proteins, reduced serum half-life, or increased serum half-life, as compared to an intact, unaltered antibody having about the same immunogenicity. For example, certain antibodies for use in the diagnostic and therapeutic methods described herein are domain deleted antibodies comprising a polypeptide chain similar to an immunoglobulin heavy chain, but lacking at least a portion of one or more heavy chain domains. For example, in certain antibodies, an entire domain of the constant region of the modified antibody is deleted, e.g., all or a portion of the CH2 domain will be deleted. In other embodiments, certain antibodies useful in the diagnostic and therapeutic methods described herein have a constant region, such as an IgG heavy chain constant region, that is altered to eliminate glycosylation, referred to elsewhere herein as an aglycosylated or "agly" antibody. Such "agly" antibodies can be made enzymatically as well as by engineering consensus glycosylation sites in the constant region. Without being bound by theory, it is believed that "agly" antibodies may have improved safety and stability characteristics in vivo. Methods for producing aglycosylated antibodies with desired effector functions are found, for example, in international application WO2005/018572, which is incorporated herein by reference in its entirety.

In certain antibodies, or DPR-binding fragments, variants, or derivatives thereof, described herein, the Fc portion can be mutated to reduce effector function using techniques known in the art. For example, deletion or inactivation (by point mutation or otherwise) of the constant region domain may decrease Fc receptor binding of the circulating modified antibody, thereby increasing DPR protein localization. In other cases, it may be that, consistent with the present invention, the constant region modification may attenuate complement binding and thus reduce the serum half-life and non-specific association of the conjugated cytotoxin. In addition, other modifications of the constant region can be used to modify disulfide bonds or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of the modification, such as DPR protein localization, biodistribution and serum half-life, can be readily measured and quantified using well-known immunological techniques without undue experimentation.

In certain antibodies, or DPR-binding fragments, variants or derivatives thereof, described herein, the Fc portion may be mutated or exchanged for an alternative protein sequence to increase cellular uptake of the antibody, for example by enhancing receptor-mediated endocytosis of the antibody via the Fc γ receptor, LRP or Thy1 receptor, or by the "SuperAntibody technique" which is said to shuttle the antibody into living cells without harming them (Expert opin. biol. ther. (2005), 237-241). For example, fusion proteins of antibody binding regions and cell surface receptors or the generation of cognate protein ligands for bispecific or multispecific antibodies with specific sequences that bind to DPRs and cell surface receptors can be engineered using techniques known in the art.

In certain antibodies, or DPR-binding fragments, variants, or derivatives thereof, described herein, the Fc portion may be mutated or exchanged for an alternative protein sequence, or the antibody may be chemically modified to increase its blood-brain barrier penetration.

Modified forms of the antibodies of the invention, or DPR-binding fragments, variants or derivatives thereof, may be prepared from intact precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein. The antibodies, or antigen-binding fragments, variants, or derivatives thereof, of the invention can be prepared or made using techniques known in the art. In certain embodiments, the antibody molecule or fragment thereof is "recombinantly produced," i.e., produced using recombinant DNA techniques. Exemplary techniques for preparing antibody molecules or fragments thereof are discussed in more detail elsewhere herein.

The antibodies or DPR-binding fragments, biotechnological variants or derivatives thereof of the invention also include derivatives that are modified, for example by covalently linking any type of molecule to the antibody, such that the covalent linkage does not prevent the antibody from specifically binding to its cognate epitope. For example, but not limited to, antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. Any of a variety of chemical modifications can be made by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more non-canonical amino acids.

Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, Fab and F (ab')₂Fragments may be produced recombinantly or by using enzymes such as papain (for producing Fab fragments) or pepsin (for producing F (ab')₂Fragments) of the immunoglobulin molecule. F (ab')₂Fragments contain the variable region, the light chain constant region, and the CH1 domain of the heavy chain. Such fragments are sufficient for use, for example, in immunodiagnostic procedures involving coupling of immunospecific portions of immunoglobulins to detection reagents such as radioisotopes.

The antibodies of the invention may be produced by any method known in the art for antibody synthesis, in particular by chemical synthesis or optionally by recombinant expression techniques as described herein.

In one embodiment, an antibody of the invention, or a DPR-binding fragment, variant or derivative thereof, comprises a synthetic constant region in which one or more domains are partially or completely deleted ("domain deleted antibody"). In certain embodiments, a compatibly-modified antibody will comprise a domain deleted construct or variant in which the entire CH2 domain has been removed (a Δ CH2 construct). For other embodiments, the deleted domains may be replaced with short linking peptides to provide flexibility and freedom of movement for the variable regions. Those skilled in the art will appreciate that such constructs are particularly preferred due to the regulatory nature of the CH2 domain on the catabolic rate of the antibody. Domain deleted constructs may use encoding IgG ₁Vectors for human constant domains are available, see for example international applications WO 02/060955 and WO 02/096948a 2. This vector was engineered to delete the CH2 domain and provide for the expression of domain deleted IgG₁Synthetic support for constant region.

In certain embodiments, the antibodies of the invention, or DPR-binding fragments, variants, or derivatives thereof, are minibodies. Minibodies can be prepared using methods described in the art, see, for example, U.S. patent 5,837,821 or international application WO 94/09817.

In one embodiment, the antibody of the invention, or a DPR-binding fragment, variant or derivative thereof, comprises an immunoglobulin heavy chain with deletions or substitutions of several or even a single amino acid, as long as it allows association between the monomeric subunits. For example, mutating a single amino acid in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding and thus increase DPR protein localization. Similarly, it may be desirable to delete only that portion of one or more constant region domains that control the effector function (e.g., complement fixation) to be modulated. Deletion of such portions of the constant region may improve selected characteristics of the antibody (serum half-life) while leaving other desired functions associated with the integrity of the constant region domain unchanged. Furthermore, as mentioned above, the constant regions of the disclosed antibodies can be synthesized by mutation or substitution of one or more amino acids that enhance the characteristics of the resulting construct. In this regard, it may be feasible to disrupt the activity provided by the conserved binding sites (e.g., Fc binding), but substantially preserve the conformational and immunogenic characteristics of the modified antibody. Other embodiments include the addition of one or more amino acids to the constant region in order to enhance desired properties such as effector function or to provide more cytotoxic or carbohydrate linkages. In such embodiments, it may be desirable to insert or replicate specific sequences derived from selected constant region domains.

Polynucleotides encoding the antibodies of the invention

The present invention also relates to one or more polynucleotides encoding any of the antibodies described in section II above, or DPR-binding fragments, variants, or derivatives thereof. The polynucleotide encoding the antibody or DPR-binding fragment, variant or derivative thereof may consist of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide encoding an antibody or a DPR-binding fragment, variant or derivative thereof may be composed of single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single-and double-stranded RNA, and RNA that is a mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-and double-stranded regions. In addition, the polynucleotide encoding the antibody or DPR-binding fragment, variant or derivative thereof may consist of a triple-stranded region comprising RNA or DNA or both RNA and DNA. Polynucleotides encoding an antibody or DPR-binding fragment, variant or derivative thereof may also contain one or more modified bases or DNA or RNA backbones modified for stability or other reasons. "modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically or metabolically modified forms.

An isolated polynucleotide encoding a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or light chain portion) can be produced by: one or more nucleotide substitutions, additions or deletions are introduced into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Optionally, conservative amino acid substitutions are made at one or more non-essential amino acid residues.

It is well known that RNA can be isolated from primary B cells, hybridoma cells or other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. If desired, mRNA can be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose. Suitable techniques are well known in the art. In one embodiment, the cdnas encoding the light and heavy chains of the antibody may be prepared simultaneously or separately using reverse transcriptase and DNA polymerase according to well-known methods. PCR can be initiated by consensus constant region primers or more specific primers based on the disclosed heavy and light chain DNA and amino acid sequences. As described above, PCR can also be used to isolate DNA clones encoding the antibody light and heavy chains. In this case, the library can be screened by consensus primers or larger homologous probes (e.g., human constant region probes).

DNA, typically plasmid DNA, can be isolated from cells using techniques known in the art, restriction mapped and sequenced according to standard, well known standard techniques, e.g., in the aforementioned references relating to recombinant DNA technology. Of course, according to the present invention, DNA may be synthesized at any time during the isolation process or subsequent analysis. The invention herein also relates to a polynucleotide encoding at least the binding domain or variable region of an immunoglobulin chain of an antibody of the invention.

In a preferred embodiment of the invention, the polynucleotide comprises a V with an anti-DPR antibody as shown in Table 2_HOr V_LA nucleic acid of the polynucleotide sequence of a region, consisting essentially of or consisting of said nucleic acid. In this regard, one skilled in the art will readily appreciate that a polynucleotide encoding at least the variable domains of the light and/or heavy chains may encode the variable domains of both immunoglobulin chains or only one. Thus, in one embodiment, the polynucleotide comprises a V having an anti-DPR antibody and/or fragment thereof as set forth in table 2_HAnd V_LA nucleic acid of the polynucleotide sequence of a region, consisting essentially of or consisting of said nucleic acid.

Table 2: v of antibodies and antibody variants recognizing poly-GA DPR, optionally C9orf72- (poly-GA) -DPR_HAnd V_LThe nucleotide sequence of the region.

The amino acid sequence of the DPR Ab-1 antibody or fragment thereof can be encoded by various nucleotide sequences. For example, codons can be optimized to maximize expression of the polypeptide. An exemplary set of nucleotide sequences encoding DPR Ab-1 is shown in table 13 below.

TABLE 13 nucleotide sequence encoding DPR Ab-1

As described elsewhere, the invention also includes fragments of the polynucleotides of the invention. In addition, polynucleotides encoding fusion polynucleotides, Fab fragments, and other biotechnological derivatives as described herein are also contemplated by the present invention.

In one embodiment, the present invention relates to a polynucleotide, optionally linked to a heterologous nucleic acid, wherein (i) said polynucleotide encodes an immunoglobulin variable heavy chain having VH-CDRs as defined in any one of the preceding clauses [1] to [10], and wherein the polynucleotide encodes a polypeptide having a VH-CDR sequence as defined in any one of the preceding clauses [1] to [10], and wherein the polynucleotide, when compared to a polynucleotide comprising the sequence of SEQ ID NO: 7 or 24, which exhibits the binding characteristics of the subject antibody as described in the examples and as described in any of the preceding clauses [1] to [36], and/or (ii) which encodes an immunoglobulin variable light chain having VL-CDRs as defined in any of the preceding clauses [1] to [10], and wherein, when paired with an immunoglobulin variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 2. 12, 15, 18 or 21, which exhibits the binding properties of the subject antibody as described in the examples and as described in any of the preceding clauses [1] to [36 ].

Furthermore, the invention relates to one or more vectors comprising one or more of those polynucleotides, optionally wherein the vector is an expression vector and the one or more polynucleotides are operably linked to an expression control sequence. Furthermore, the present invention relates to a host cell comprising one or more polynucleotides or vectors of the present invention; and a method of producing an anti-poly- (GA) -DPR antibody or DPR-binding fragment thereof, the method comprising culturing a host cell of the invention under conditions that allow expression of the anti-DPR antibody or DPR-binding fragment thereof; and isolating the anti-DPR antibody or DPR-binding fragment thereof from the culture.

Polynucleotides may be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of an antibody is known, polynucleotides encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al, BioTechniques 17(1994), 242), which, briefly, involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody or DPR-binding fragment, variant or derivative thereof may be produced from a nucleic acid from a suitable source. If clones containing nucleic acid encoding a particular antibody are not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the antibody may be obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing the antibody, such as B cells selected to express the antibody, or nucleic acid isolated therefrom, optionally poly A + RNA) by chemical synthesis, or by PCR amplification using synthetic primers that hybridize to the 3 'and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence, in order to identify cDNA clones encoding the antibody from the cDNA library. The amplified nucleic acid produced by PCR may then be cloned into a replicable cloning vector using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of an antibody or DPR-binding fragment, variant or derivative thereof has been determined, the nucleotide sequence of the antibody can be manipulated to produce antibodies having different amino acid sequences, e.g., to produce amino acid substitutions, deletions and/or insertions, using methods well known in the art for manipulating nucleotide sequences, e.g., recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, e.g., the techniques described in Sambrook et al, Molecular Cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al, eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998), which are incorporated herein by reference).

Expression of the antibody Polypeptides of the invention

After manipulation of the isolated genetic material to provide an antibody of the invention, or a DPR-binding fragment, variant or derivative thereof, the polynucleotide encoding the antibody is typically inserted into an expression vector for introduction into a host cell, which can be used to produce a desired amount of the antibody. Described herein is recombinant expression of an antibody or fragment, derivative or analogue thereof, e.g., a heavy or light chain of an antibody that binds to a target molecule. Once a polynucleotide encoding an antibody molecule of the invention, or a heavy or light chain of an antibody, or a portion thereof (optionally containing a heavy or light chain variable domain) has been obtained, vectors for producing the antibody molecule can be produced by recombinant DNA techniques using techniques well known in the art. Thus, described herein are methods for producing proteins by expressing polynucleotides comprising antibody-encoding nucleotide sequences. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. Accordingly, the invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. The vector may comprise nucleotide sequences encoding the constant regions of the antibody molecule (see, e.g., international applications WO 86/05807 and WO 89/01036; and U.S. Pat. No. 5,122,464), and the variable domains of the antibody may be cloned into such a vector to express the complete heavy chain or the complete light chain.

The term "vector" or "expression vector" as used herein means a vector used according to the present invention as a vehicle for introduction into a host cell and expression of a desired gene in said host cell. Such vectors may be readily selected from the group consisting of plasmids, phages, viruses and retroviruses, as known to those skilled in the art. Generally, vectors compatible with the present invention will contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells. For the purposes of the present invention, a variety of expression vector systems may be employed. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV), or SV40 virus. Other expression vectors involve the use of polycistronic systems with internal ribosome binding sites. Furthermore, cells that integrate DNA into their chromosomes can be selected by introducing one or more markers that allow for selection of transfected host cells. The marker may provide prototrophy, resistance to a biological agent (e.g., an antibiotic), or resistance to a heavy metal such as copper to an auxotrophic host. The selectable marker gene may be linked directly to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Other elements may also be required for optimal synthesis of mRNA. These elements may include signal sequences, splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

In a particularly preferred embodiment, the cloned variable region genes are inserted into an expression vector together with the heavy and light chain constant region genes (optionally human), as described above. In one embodiment, this is accomplished using a proprietary expression vector from Biogens, inc (known as nespla) and disclosed in U.S. patent No. 6,159,730. The vector contains a cytomegalovirus promoter/enhancer, a mouse beta globin major promoter, an SV40 origin of replication, a bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, a dihydrofolate reductase gene, and a leader sequence. This vector has been found to produce a very high degree of antibody expression after incorporation of variable and constant region genes, transfection in CHO cells, followed by selection in G418 containing medium and amplification of methotrexate. Of course, any expression vector capable of expression in eukaryotic cells can be used in the present invention. Examples of suitable vectors include, but are not limited to, plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, Calif.), and plasmid PCI (available from Promega, Madison, Wis.). In general, screening of large numbers of transformed cells for those expressing suitably higher levels of immunoglobulin heavy and light chains is a routine experiment that can be performed, for example, by a robotic system. Vector systems are also taught in U.S. Pat. nos. 5,736,137 and 5,658,570, each of which is incorporated by reference in its entirety. This system provides high expression levels, e.g., > 30 Pg/cell/day. Other exemplary expression vector systems are disclosed, for example, in U.S. patent No. 6,413,777.

In other preferred embodiments, the antibodies of the invention, or DPR-binding fragments, variants or derivatives thereof, may be expressed using polycistronic constructs, such as those disclosed in U.S. patent application publication No. 2003-0157641 a1, and which patents are incorporated herein in their entirety. In these expression systems, multiple gene products of interest, such as the heavy and light chains of an antibody, can be produced from a single polycistronic construct. These systems advantageously use an Internal Ribosome Entry Site (IRES) to provide relatively high levels of antibodies. Suitable IRES sequences are disclosed in U.S. patent No. 6,193,980, which is also incorporated herein. One skilled in the art will appreciate that such expression systems can be used to efficiently produce all of the antibodies disclosed herein. Thus, in one embodiment, the invention provides a vector comprising a polynucleotide encoding at least the binding domain or variable region of an immunoglobulin chain of an antibody, optionally in combination with a polynucleotide encoding the variable region of another immunoglobulin chain of the binding molecule.

More generally, once a vector or DNA sequence encoding a monomeric subunit of an antibody has been prepared, the expression vector can be introduced into an appropriate host cell. Introduction of plasmids into a host In the host cell can be achieved by various techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including lipofection, using, for exampleOr lipofectamine), protoplast fusion, calcium phosphate precipitation, fusion of cells with enveloped DNA, microinjection, and whole virus infection. Typically, the plasmid is introduced into the host via standard calcium phosphate co-precipitation methods. Host cells with the expression constructs were grown under conditions suitable for production of light and heavy chains, and heavy and/or light chain protein synthesis was assayed. Exemplary analytical techniques include enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), or fluorescence activated cell sorting analysis (FACS), immunohistochemistry, and the like.

The expression vector is transferred to a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibody for use in the methods described herein. Thus, the invention includes a host cell comprising a polynucleotide encoding an antibody of the invention or a heavy or light chain thereof, or at least an immunoglobulin binding domain or variable region thereof, optionally operably linked to a heterologous promoter. Additionally or alternatively, the invention also includes a host cell comprising a vector as defined above comprising a polynucleotide encoding at least the binding domain or the variable region of the immunoglobulin chain of the antibody, optionally in combination with a polynucleotide encoding the variable region of another immunoglobulin chain of the binding molecule. In a preferred embodiment of expressing a diabody, a single vector or multiple vectors encoding the heavy and light chains can be co-expressed in a host cell for expression of the complete immunoglobulin molecule, as described in detail below.

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain-derived polypeptide and the second vector encoding a light chain-derived polypeptide. Both vectors may contain the same selectable marker, allowing for equal expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding both the heavy and light chain polypeptides may be used. In such cases, it is advantageous to place the light chain before the heavy chain in order to avoid an excess of non-toxic heavy chains; see Proudfoot, Nature 322(1986), 52; kohler, Proc.Natl.Acad.Sci.USA 77(1980), 2197. The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

As used herein, "host cell" refers to a cell containing a vector constructed using recombinant DNA techniques and encoding at least one heterologous gene. In the description of the methods for isolating antibodies from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to indicate the source of the antibody unless clearly indicated otherwise. In other words, recovery of the polypeptide from "cells" can mean recovery from whole cells separated by centrifugation or from a cell culture containing media and suspended cells.

A variety of host expression vector systems may be used to express the antibody molecules for use in the methods described herein. Such host expression systems represent vehicles by which the coding sequence of interest can be produced and subsequently purified, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequence, express the antibody molecules of the invention in situ. These host expression systems include, but are not limited to: microorganisms, such as bacteria (e.g., E.coli, Bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., saccharomyces, pichia) transformed with a recombinant yeast expression vector containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant viral expression vectors containing antibody coding sequences (e.g., cauliflower mosaic virus (CaMV); Tobacco Mosaic Virus (TMV)) or transformed with recombinant plasmid expression vectors containing antibody coding sequences (e.g., Ti plasmid); or mammalian cell systems (e.g., COS, CHO, NSO, BLK, 293, 3T3 cells) containing recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinovirus 7.5K promoter). Optionally, bacterial cells (e.g., E.coli) are used for expression of the recombinant antibody molecule, or optionally eukaryotic cells, particularly where the recombinant antibody molecule is expressed intact. For example, mammalian cells (e.g., chinese hamster ovary Cells (CHO)) in combination with vectors (e.g., major mid-early gene promoter elements from human megavirus) are efficient expression systems for antibodies; see, e.g., Foecking et al, Gene 45(1986), 101; cockett et al, Bio/Technology 8(1990), 2.

Host cell lines for protein expression are often of mammalian origin; it is believed that it is within the ability of one skilled in the art to preferentially determine the particular host cell line that is most suitable for expression of the desired gene product therein. Exemplary host cell lines include, but are not limited to, CHO (chinese hamster ovary), DG44 and DUXB11 (chinese hamster ovary cell line, DHFR minus), HELA (human cervical cancer), CVI (monkey kidney cell line), COS (derivative of CVI with SV 40T antigen), VERY, BHK (baby hamster kidney), MDCK, WI38, R1610 (chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney cell line), SP2/O (mouse myeloma), P3x63-ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), and 293 (human kidney). CHO and 293 cell lines are particularly preferred. Host cell lines are generally available from commercial services, the American Tissue Culture Collection (American Tissue Culture Collection) or from the open literature.

In addition, host cell strains may be selected that modulate the expression of the inserted sequences or modify and process the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important with respect to protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems may be selected to ensure proper modification and processing of the expressed foreign protein. To this end, eukaryotic host cells with the cellular machinery (cellular machinery) for appropriate processing of the primary transcript, glycosylation of the gene product, and phosphorylation can be used.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines stably expressing the antibody molecule can be engineered. Instead of using an expression vector containing a viral origin of replication, a host cell can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introduction of the exogenous DNA, the engineered cells can be allowed to grow in enriched media for 1-2 days and then switched to selective media. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form loci, which in turn can be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that stably express the antibody molecule.

A variety of selection systems can be used, including but not limited to herpes simplex virus thymidine kinase (Wigler et al, Cell 11(1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48(1992), 202) and adenine phosphoribosyltransferase (Lowy et al, Cell 22(1980), 817) genes can be used in tk-, hgprt-or aprt-cells, respectively. In addition, antimetabolite resistance can be used as a basis for selecting the following genes: dhfr (Wigler et al, Natl.Acad.Sci.USA 77(1980), 357; O' Hare et al, Proc.Natl.Acad.Sci.USA 78(1981), 1527) which confers resistance to methotrexate; gpt conferring mycophenolic acid resistance (Mulligan and Berg, proc.natl.acad.sci.usa 78(1981), 2072); neo (Goldspir et al, Clinical Pharmacy 12(1993), 488-42; Wu and Wu, Biotherapy 3(1991), 87-95; Tolstoshiev, Ann. Rev. Pharmacol. Toxicol.32(1993), 573-596; Mulligan, Science 260(1993), 926-932; and Morgan and Anderson, Ann. Rev. biochem.62(1993), 191-217; TIB TECH 11(1993), 155-215;); and hygro (Santerre et al, Gene 30(1984), 147) which confers resistance to hygromycin. Methods generally known in the art of recombinant DNA technology that can be used are described in the following documents: ausubel et al (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and chapters 12 and 13, Dracopoli et al (ed.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al, J.mol.biol.150: 1(1981), the entirety of which is incorporated herein by reference. Expression levels of antibodies can be increased by vector amplification, for a review see Bebbington and Hentschel, The use of vector based on gene amplification for The expression of bound genes in mammalian cells in DNA cloning, Academic Press, New York, Vol.3 (1987). When the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the host cell culture will result in an increase in the copy number of the marker gene. Since the amplified region is associated with an antibody gene, antibody production will also increase; see Crouse et al, mol.cell.biol.3(1983), 257.

In vitro production may allow scale-up to obtain large quantities of the desired antibody. Techniques for mammalian culture under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g., in an airlift reactor or in a continuous stirred reactor; or fixed or entrapped cell culture, for example in hollow fibers, microcapsules, on agarose beads or ceramic cartridges. If necessary and/or desired, the solution of the polypeptide can be purified by customary chromatographic methods, for example gel filtration, ion exchange chromatography, chromatography via DEAE-cellulose chromatography or (immuno) affinity chromatography, for example after the preferred biosynthesis of the synthetic hinge region polypeptide or before or after the HIC chromatography step described herein.

The gene encoding the antibody of the invention or a DPR-binding fragment, variant or derivative thereof may also be expressed in non-mammalian cells such as bacterial or insect or yeast or plant cells. Bacteria that readily take up nucleic acids include members of the following: strains of enterobacteriaceae, such as escherichia coli or salmonella; bacillaceae, such as bacillus subtilis; pneumococcus; streptococcus, and Haemophilus influenzae. It is further understood that when expressed in bacteria, the heterologous polypeptide typically becomes part of an inclusion body. The heterologous polypeptide must be isolated, purified and then assembled into a functional molecule. When a tetravalent form of the antibody is desired, the subunits are then self-assembled into a tetravalent antibody; see, for example, international application WO 02/096948.

In bacterial systems, a number of expression vectors are suitably selected depending on the intended use of the expressed antibody molecule. For example, when large quantities of the protein are to be produced to produce pharmaceutical compositions of antibody molecules, vectors directing the expression of high levels of fusion protein products that are easy to purify may be desirable. Such vectors include, but are not limited to: coli expression vector pUR278(Ruther et al, EMBO j.2(1983), 1791) in which the antibody coding sequence can be separately ligated into the vector in-frame with the lacZ coding region in order to produce a fusion protein; pIN vectors (Inouye and Inouye, Nucleic Acids Res.13(1985), 3101-; and so on. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads, followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned gene product of interest can be released from the GST moiety.

In addition to prokaryotes, eukaryotic microorganisms may also be used. Saccharomyces cerevisiae or common baker's yeast are the most commonly used in eukaryotic microorganisms, although many other strains are commonly available, e.g., Pichia pastoris. For expression in Saccharomyces, for example, the plasmid YRp7(Stinchcomb et al, Nature 282(1979), 39; Kingsman et al, Gene 7(1979), 141; Tschemper et al, Gene 10(1980), 157) is commonly used. This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutant strains lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85(1977), 12). The presence of trpl lesion, characteristic of the yeast host cell genome, then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is generally used as a vector for expressing foreign genes. The virus was grown in Spodoptera frugiperda (Spodoptera frugiperda) cells. Antibody coding sequences can be cloned separately into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

Once the antibody molecules of the invention have been recombinantly expressed, the intact antibodies of the invention, dimers, single light and heavy chains thereof, or other immunoglobulin forms thereof, can be purified according to standard procedures in the art, including, for example, by: chromatography (e.g., ion exchange, affinity, particularly affinity for a particular antigen after passage through protein a, and size column chromatography), centrifugation, differential solubility (e.g., ammonium sulfate precipitation), or by any other standard technique for purifying proteins; see, e.g., Scopes, "Protein Purification", Springer Verlag, n.y. (1982). Alternatively, preferred methods for increasing the affinity of the antibodies of the invention are disclosed in U.S. patent publication 2002-0123057A 1. Thus, in one embodiment, the invention also provides a method of making an anti-DPR antibody or an antibody recognizing a mutated and/or aggregated C9orf72-DPR species and/or fragments thereof or immunoglobulin chains thereof, the method comprising:

(a) culturing a host cell as defined above, said cell comprising a polynucleotide or vector as defined above; and

(b) isolating the antibody or immunoglobulin chain thereof from the culture.

Furthermore, the present invention also relates to an antibody or immunoglobulin chain thereof encoded by a polynucleotide as defined above or obtainable by said method for preparing an anti-DPR antibody or an antibody recognizing a mutated and/or aggregated C9orf72-DPR species and/or a fragment thereof or immunoglobulin chain thereof.

Fusion proteins and conjugates of the invention

In certain embodiments, an antibody polypeptide comprises an amino acid sequence or one or more portions that are not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, single chain Fv antibody fragments of the invention may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, drug, toxin, or label, such as fluorescent, radioactive, enzymatic, nuclear magnetic, heavy metal, etc.).

The antibody polypeptides of the invention may comprise, consist essentially of, or consist of a fusion protein. Fusion proteins are chimeric molecules comprising, for example, an immunoglobulin DPR binding domain having at least one target binding site and at least one heterologous moiety (i.e., a moiety not naturally linked in nature). These amino acid sequences may typically be present in separate proteins, they may be combined together in a fusion polypeptide, or the polypeptides may typically be present in the same protein but placed in a new arrangement in a fusion polypeptide. Fusion proteins can be produced, for example, by chemical synthesis, or by generating and translating polynucleotides in which peptide regions are encoded in the desired relationship.

The term "heterologous" as applied to a polynucleotide or polypeptide means that the polynucleotide or polypeptide is derived from an entity that is genotypically different from the rest of the entity to which it is compared. For example, as used herein, a "heterologous polypeptide" fused to an antibody or antigen-binding fragment, variant, or analog thereof is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide of a different species.

As discussed in more detail elsewhere herein, the antibodies of the invention or DPR-binding fragments, variants or derivatives thereof can be further recombinantly fused at the N-or C-terminus to a heterologous polypeptide or chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide or other composition. For example, the antibody may be recombinantly fused or conjugated to molecules and effector molecules that serve as labels in detection assays, such as heterologous polypeptides, drugs, radionuclides, or toxins; see, for example, international application WO 92/08495; WO 91/14438; WO 89/12624; U.S. patent nos. 5,314,995; and european patent application EP 0396387.

The antibody of the invention or a DPR-binding fragment, variant or derivative thereof may consist of amino acids linked to each other by peptide bonds or modified peptide bonds (i.e. peptide isosteres) and may contain amino acids other than the 20 gene-encoded amino acids. Antibodies can be modified by natural processes (e.g., post-translational processing) or by chemical modification techniques well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in the antibody, including the peptide backbone, amino acid side chains, and amino or carboxyl termini, or on moieties such as carbohydrates. It is understood that the same type of modification may be present to the same or different extent at several sites of a given antibody. In addition, a given antibody may contain many types of modifications. Antibodies may be branched, for example due to ubiquitination, and they may be cyclic, with or without branching. The cyclic, branched and branched cyclic antibodies can be produced by post-translational natural processes or can be prepared by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamic acid, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenization, sulfation, transfer RNA mediated addition of amino acids to proteins (e.g., arginylation), and ubiquitination; see, e.g., protein-Structure And Molecular Properties, t.e. creatton, w.h.freeman And Company, New York 2 edition (1993); posttranslational relative Modification Of Proteins, b.c. johnson, editors, Academic Press, New York, (1983) 1-12; seifter et al, meth.enzymol.182(1990), 626-; rattan et al, Ann.NY Acad.Sci.663(1992), 48-62).

As discussed elsewhere herein, an antibody of the invention or a DPR-binding fragment, variant or derivative thereof can be fused to a heterologous polypeptide to increase the in vivo half-life of the polypeptide or used in immunoassays using methods known in the art. For example, in one embodiment, PEG may be conjugated to antibodies of the invention to increase their half-life in vivo; see, e.g., Leong et al, Cytokine 16(2001), 106-; in Drug deliv.rev.54(2002), 531; or Weir et al, biochem.

Furthermore, the antibodies of the invention or DPR-binding fragments, synthetic variants or biotechnological derivatives thereof may be fused to a marker sequence (e.g. a peptide) to facilitate their purification or detection. In a preferred embodiment, the marker amino acid sequence is hexa-histidine peptide (HIS) (SEQ ID NO: 84), such as the tags provided in pQE vector (QIAGEN, Inc., 9259Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. For example, hexahistidine provides convenient purification of the fusion protein as described in Gentz et al, Proc.Natl.Acad.Sci.USA 86(1989), 821-824. Other peptide tags that may be used for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from influenza hemagglutinin protein (Wilson et al, Cell 37(1984), 767), GST, c-myc, and the "flag" tag; see, e.g., Bill Brizzard, BioTechniques 44(2008) 693-.

Fusion proteins can be prepared using methods well known in the art; see, for example, U.S. Pat. nos. 5,116,964 and 5,225,538. The precise site at which the fusion is performed can be selected empirically to optimize the secretion or binding properties of the fusion protein. The DNA encoding the fusion protein is then transfected into a host cell for expression, which is performed as previously described herein.

The antibodies of the invention may be used in unconjugated form or may be conjugated to at least one of a variety of molecules, for example, to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging or therapy of a patient. When purified, the antibodies of the invention or DPR-binding fragments, variants or derivatives thereof may be labeled or conjugated before or after purification. In particular, an antibody of the invention, or a DPR-binding fragment, variant or derivative thereof, may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, agent, or PEG.

One skilled in the art will appreciate that conjugates can also be assembled using a variety of techniques, depending on the agent selected to be conjugated. For example, conjugates with biotin are prepared, for example, by reacting a DPR-binding polypeptide with an activated ester of biotin, such as biotin N-hydroxysuccinimide ester. Similarly, conjugates with fluorescent labels can be prepared in the presence of a coupling agent (such as those listed herein) or by reaction with an isothiocyanate, optionally fluorescein-isothiocyanate. Conjugates of the antibodies of the invention or DPR-binding fragments, variants or derivatives thereof are prepared in a similar manner.

The invention also includes an antibody of the invention, or a DPR-binding fragment, variant or derivative thereof, conjugated to a diagnostic or therapeutic agent. The antibodies are useful in diagnosis, e.g., demonstrating the presence of DPR to indicate a risk of developing a disease or disorder associated with DPR, optionally associated with C9orf72 mutated to form DPR, i.e., C9orf72-DPR, to monitor the development or progression of such diseases, i.e., to show the occurrence of or associated with DPR or aggregated forms thereof, or as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment and/or prevention regimen. Thus, in one embodiment, the invention relates to an antibody that is detectably labeled. Furthermore, in one embodiment, the invention relates to an antibody linked to a drug. Detection may be facilitated by coupling the antibody or DPR-binding fragment, variant or derivative thereof to a detectable substance. The detectable substance or label may typically be an enzyme; a heavy metal, optionally gold; a dye, optionally a fluorescent or luminescent dye; or a radioactive label. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emission using various positron emission tomography Metal and non-radioactive paramagnetic metal ions; see, for example, U.S. Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostic agents according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of the bioluminescent material include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include¹²⁵I、¹³¹I、¹¹¹In or⁹⁹Tc. Accordingly, in one embodiment, the invention provides a detectably labeled antibody, wherein the detectable label is selected from the group consisting of: enzymes, radioisotopes, fluorophores, and heavy metals.

The antibody or DPR-binding fragment, variant or derivative thereof may also be detectably labeled by coupling to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence generated during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

One way in which an antibody or DPR-binding fragment, variant or derivative thereof may be detectably labeled is by linking it to an Enzyme and using The Linked product in an Enzyme Immunoassay (EIA) (Voller, a., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates quick Publication, walker, Md., Diagnostic Horizons 2(1978), 1-7); voller et al, J.Clin.Pathol.31(1978), 507- & 520; butler, meth.enzymol.73(1981), 482-; maggio, (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980); ishikawa, et al, (ed.), Enzyme Immunoassay, Kgaku Shoin Tokyo (1981). The enzyme bound to the antibody will react with a suitable substrate (optionally a chromogenic substrate) to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric, or visual means. Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Alternatively, the detection may be accomplished by colorimetric methods that employ enzymes to obtain chromogenic substrates. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of the substrate as compared to a similarly prepared standard.

Detection can also be accomplished using any of a variety of other immunoassays. For example, by radiolabelling an antibody or a DPR-binding fragment, variant or derivative thereof, it is possible to detect The antibody by using Radioimmunoassay (RIA) (see, e.g., Weintraub, b., Principles of radioimmunoassay, seven Training couse on radio ligand and Assay technologies, The Endocrine Society, (3 months 1986)), which is incorporated herein by reference). The radioisotope may be detected by means including, but not limited to, gamma counter, scintillation counter, or autoradiography.

Antibodies or DPR-binding fragments, variants or derivatives thereof may also be used with metals that emit fluorescence such as¹⁵²Eu or other metals of the lanthanide series. Such metal chelating groups, such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA), can be used to attach these metals to antibodies.

Techniques For conjugating various moieties to Antibodies or DPR-binding fragments, variants or derivatives thereof are well known, see, For example, Arnon et al, "Monoclonal Antibodies For immunological targeting Of Drugs In Cancer Therapy", In Monoclonal Antibodies And Cancer Therapy, Reisfeld et al (ed.), pages 243-56 (Alan R.Liss, Inc. (1985); Hellstrom et al, "Antibodies For Drug Delivery Therapy", In Controlled Drug Delivery Therapy (2 nd edition), Robinson et al (ed.), mark Delivery Therapy, (1987) Delivery 53; Thorpe, "Antibodies Of Cytotoxic Agents In Therapy;" Monoclonal Antibodies In Therapy, "Biological Delivery Therapy," Biological assay, "filtration assay In Therapy, filtration assay, repair, molecular Therapy, protein, baldwin et al (eds.), Academic Press (1985)303-16 And Thorpe et al, "The recommendation And cytoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev.62(1982), 119-.

As mentioned, in certain embodiments, moieties that enhance the stability or efficacy of binding molecules, e.g., binding polypeptides, e.g., antibodies or immunospecific fragments thereof, may be conjugated. For example, in one embodiment, PEG may be conjugated to a binding molecule of the invention to increase its half-life in vivo. Leong et al, Cytokine 16(2001), 106; in Drug deliv.rev.54(2002), 531; or Weir et al, biochem.

Compositions and methods of use of the invention

The present invention relates to a composition comprising a DPR binding molecule of the invention as described above, e.g. an antibody or a DPR binding fragment, variant or biotechnological derivative thereof, or a polynucleotide, vector or cell of the invention, as defined above. In one embodiment, the composition of the invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier. Furthermore, depending on the intended use of the pharmaceutical composition, the pharmaceutical composition of the present disclosure may comprise other agents, such as an interleukin or an interferon. For use in the treatment of a disease or condition (such as FTLD) which shows the occurrence of or is associated with DPR or aggregated forms thereof (in particular C9orf72-DPR), the further agent may be selected from the group consisting of: small organic molecules, anti-DPR antibodies, and combinations thereof. Thus, in a particularly preferred embodiment, the invention relates to the use of a DPR binding molecule, e.g. an antibody of the invention or a DPR binding fragment thereof or a binding molecule having substantially the same binding specificity as any one thereof, a polynucleotide, a vector or a cell of the invention for the preparation of a pharmaceutical or diagnostic composition for the prophylactic and therapeutic treatment of a disease or disorder associated with a DPR protein, for monitoring the progression of a disease or disorder associated with a DPR protein and/or aggregated C9orf72 or for the response of a subject to a DPR treatment or for determining the risk of a subject to develop a disease or disorder associated with a DPR protein and/or aggregated C9orf 72-DPR.

Accordingly, in one embodiment, the invention relates to a method of treating a disease or disorder characterized by abnormal accumulation and/or deposition of DPR and DPR proteins, such as aggregated C9orf72 due to C9orf72-DPR, comprising administering to a subject in need thereof a therapeutically effective amount of any of the aforementioned DPR binding molecules, antibodies, polynucleotides, vectors or cells of the invention.

A particular advantage of the treatment method of the invention lies in the fact that: the recombinant antibodies of the invention are derived from B cells or memory B cells of healthy human subjects without symptoms or signs of disease, e.g., carrying asymptomatic mutations and/or mutations, show or are associated with the development of DPR or aggregated forms thereof, and thus have the possibility of preventing clinical manifestations associated with DPR, e.g., C9orf72 with amplified hexanucleotide repeat sequence mutations, resulting in the formation of dipeptide repeats (DPR) in the C9orf72 protein and aggregated C9orf72 due to C9orf72-DPR, or reducing the risk of the development of clinical manifestations or disease or disorder or delaying the onset or progression of a clinical manifestations disease or disorder. In general, the antibodies of the invention have also successfully undergone somatic maturation, i.e., optimization of selectivity and effectiveness in high affinity binding to target DPR molecules by somatic variation of antibody variable regions.

The knowledge that such cells have not been activated by related or other physiological proteins or cellular structures in vivo, e.g. in humans for autoimmune or allergic reactions, is also of great medical importance as this means that the chances of successfully passing the clinical testing phase are greatly increased. It can be said that in at least one human subject, efficacy, acceptability and tolerance have been demonstrated prior to preclinical and clinical development of prophylactic or therapeutic antibodies. Thus, the high target structure-specific affinity of the human anti-DPR antibodies of the invention as therapeutics and their reduced potential for side effects can be expected to significantly increase their likelihood of clinical success.

The invention also provides a pharmaceutical and diagnostic package or kit comprising one or more packs filled with one or more of the above-described components, e.g., an anti-DPR antibody of the invention, a binding fragment thereof, a biotechnological derivative or variant thereof, a polynucleotide of the invention, a vector or a cell, respectively. Accompanying the container may be a report in tabular form issued by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, the report reflecting approval for human administration by the agency of manufacture, use or sale. Additionally or alternatively, the kit includes reagents and/or instructions for use in a suitable diagnostic assay. Compositions, e.g., kits of the invention, are particularly useful for risk assessment, diagnosis, prevention and treatment of diseases or conditions that are accompanied by the presence of DPR, and are particularly useful for the treatment of diseases that are generally characterized by the presence of DPR. In particular, the compositions are useful for treating disorders associated with DPR aggregation, such as C9orf72 with mutations of the amplified hexanucleotide repeat sequences, the formation of aggregated C9orf72 due to C9orf 72-DPR. Diseases and/or disorders associated with DPR include, but are not limited to, frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), FTLD-ALS, and/or spinocerebellar ataxia type 36.

The pharmaceutical compositions of the present invention may be formulated according to methods well known in the art; see, e.g., Remington: the Science and Practice of Pharmacy (2000) by The University of Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions comprising such carriers may be formulated by well-known conventional methods. These pharmaceutical compositions may be administered to a subject at a suitable dose. Administration of suitable compositions can be carried out by different means, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, topical or intradermal administration or spinal or brain delivery. Aerosol formulations, such as nasal spray formulations, include purified aqueous or other solutions of the active agent with preservatives and isotonicity agents. Such formulations are optionally adjusted to a pH and isotonic state compatible with the nasal mucosa. Formulations for rectal or vaginal administration may be presented as suppositories with suitable carriers.

The dosage regimen will be determined by the attending physician and by clinical factors. As is well known in the medical arts, the dosage for any one patient depends on many factors, including the size of the patient, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs currently being administered. Typical doses may range, for example, from 0.001 to 1000 μ g (or again ranging nucleic acid for expression or for inhibition of expression); however, doses below or above this exemplary range are contemplated, particularly in view of the factors mentioned above. Typically, the dose can range, for example, from about 0.0001mg/kg to 100mg/kg, and more typically from 0.01mg/kg to 5mg/kg (e.g., 0.02mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 1mg/kg, 2mg/kg, etc.) of the body weight of the host. For example, the dose may be 1mg/kg body weight or 10mg/kg body weight or in the range 1-10mg/kg, optionally at least 3, 10 or 30 mg/kg. Intermediate doses within the above ranges are also intended to be within the scope of the present invention. The subject may administer the dose daily, alternately by day, weekly, or according to other time courses determined by empirical analysis. Exemplary treatments require administration in multiple doses over an extended period of time (e.g., at least six months). Additional exemplary treatment regimens require administration once every two weeks or once a month or once every 3 to 6 months. Exemplary dosage regimens include 1-10mg/kg or 15mg/kg continuously or every other day, or 30mg/kg weekly; see also example 18. In some methods, two or more monoclonal antibodies with different binding specificities can be administered simultaneously, in which case the dose of each antibody administered falls within the indicated ranges. Progress can be monitored by periodic assessment. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, among others. Furthermore, depending on the intended use of the pharmaceutical composition, the pharmaceutical composition of the invention may comprise other agents, such as dopamine or psychopharmacological drugs.

Furthermore, in a preferred embodiment of the invention, the pharmaceutical composition of the invention may be formulated as a vaccine for passive immunization, e.g. if it comprises an anti-DPR antibody or a DPR binding fragment, or a synthetic or biotechnological variant or derivative thereof. As described in the background section, aggregated DPR species are the primary causes of diseases and/or conditions such as FTLD and ALS. Therefore, it is prudent to expect that passive immunization with human anti-DPR antibodies and equivalent DPR binding molecules of the invention will help avoid several adverse effects of the active immunotherapy concept and lead to a reduction in the aggregation of DPRs. Accordingly, the anti-DPR antibodies of the invention and their equivalents will be particularly useful as vaccines for preventing or ameliorating diseases or disorders (e.g., FTLD) that show the presence of or are caused by DPR or its aggregated form (particularly C9orf 72-DPR).

In one embodiment, it may be beneficial to use recombinant fab (rfab) and single chain fragments (scFv) of the antibodies of the invention, which can penetrate the cell membrane more easily. For example, Robert et al, Protein eng.des.sel. (2008); s1741-0134, published on-line in advance, describes the use of chimeric recombinant fab (rbab) and single chain fragments (scFv) of the monoclonal antibody WO-2, which recognizes an epitope in the N-terminal region of Abeta. The engineered fragments are capable of (i) preventing amyloid fibrillation, (ii) breaking down preformed Abeta1-42 fibrils, and (iii) inhibiting Abeta1-42 oligomer-mediated neurotoxicity, as effective as intact IgG molecules in vitro. Recognized advantages of using small Fab and scFv engineered antibody formats lacking effector functions include more efficient crossing of the blood-brain barrier and minimizing the risk of triggering inflammatory side reactions. Furthermore, in addition to scFv and single domain antibodies retaining the binding specificity of full-length antibodies, they can also be expressed as a single gene and intracellularly as intrabodies in mammalian cells, with the potential to alter the folding, interaction, modification or subcellular localization of their targets; for a review see, e.g., Miller and Messer, Molecular Therapy 12(2005), 394-401.

Among the different approaches, Muller et al, Expert Opin biol. Ther. (2005), 237-. This cell-penetrating antibody opens new diagnostic and therapeutic windows. The term "TransMab" was created for these antibodies.

In another embodiment, it may be desirable to co-administer or sequentially administer other antibodies that may be used to treat diseases, disorders, or symptoms associated with the occurrence of DPRs, particularly aggregated DPRs, e.g., C9orf 72-DPRs. In one embodiment, the additional antibody is comprised in a pharmaceutical composition of the invention. Examples of antibodies that may be used to treat a subject include, but are not limited to, antibodies targeting CD33, SGLT2, IL-6, and IL-1.

In another embodiment, it may be desirable to co-administer or sequentially administer other agents useful for treating diseases, disorders, or symptoms associated with DPR (particularly aggregated DPR, such as mutated C9orf72, i.e., C9orf 72-DPR). In one embodiment, an additional agent is included in the pharmaceutical composition of the invention. Examples of agents useful for treating a subject include, but are not limited to: VMAT2 inhibitors that target involuntary muscle movements, such as anti-inflammatory agents like diflunisal, corticosteroids, 2- (2, 6-dichloroanilino) phenylacetic acid (diclofenac), isobutylpropionic acid-phenolic acid (ibuprofen); diuretics, epigallocatechin gallate, melphalan hydrochloride, dexamethasone, bortezomib-melphalan, bortezomib-dexamethasone, melphalan-dexamethasone, bortezomib-melphalan-dexamethasone; antidepressants, antipsychotics, neuroleptics, antidementives (e.g., the NMDA-rezeptor antagonist memantine), acetylcholinesterase inhibitors (e.g., donepezil, hydrochloric acid, rivastigmine, galantamine), glutamate antagonists, and other nootropic hypotensives (e.g., dihydralazine, methyldopa), cytostatics, glucocorticoids, Angiotensin Converting Enzyme (ACE) inhibitors; an anti-inflammatory agent, or any combination thereof.

A therapeutically effective dose or amount refers to an amount of active ingredient sufficient to ameliorate the symptoms or condition. The therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED₅₀(dose therapeutically effective in 50% of the population) and LD₅₀(dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀。

From the foregoing, it is apparent that the present invention encompasses any use of DPR binding molecules comprising at least the CDRs and variants thereof of the above-described antibodies, particularly for diagnosing and/or treating diseases or disorders (e.g. FTLD) associated with DPR (particularly aggregated DPR species, such as C9orf 72-DPR). Optionally, the binding molecule is an antibody of the invention or a biotechnological derivative thereof.

In another embodiment, the present invention relates to a diagnostic composition comprising any of the above-described DPR binding molecules, antibodies, DPR binding fragments, polynucleotides, vectors or cells of the invention and optionally suitable detection means, such as reagents conventionally used for detection in immunological or nucleic acid based diagnostic methods. The antibodies of the invention are suitable, for example, for use in immunoassays in which the antibodies can be utilized in liquid phase or bound to a solid support. Examples of immunoassays which can utilize the antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are Radioimmunoassays (RIA), sandwich immunoassays (immunometric assays), flow cytometry and western blot assays. The antibodies of the invention can be bound to many different carriers and used to isolate cells that specifically bind thereto. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agarose, and magnetite. For the purposes of the present invention, the carrier may be soluble or insoluble in nature. Many different labels and labeling methods are known to those of ordinary skill in the art. Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds; see also the embodiments discussed above.

By another embodiment, DPR binding molecules, in particular antibodies of the invention, may also be used in a method for diagnosing a disease or disorder in an individual by obtaining a body fluid sample from the individual to be tested (which body fluid sample may be a blood sample, a plasma sample, a serum sample, a lymph sample or any other body fluid sample, such as saliva or urine sample) and contacting the body fluid sample with an antibody of the invention under conditions enabling the formation of an antibody-antigen complex. The level of such complexes is then determined by methods known in the art, a level significantly higher than the level formed in the control sample being indicative of a disease or disorder in the individual being tested. In the same manner, specific antigens bound by the antibodies of the invention may also be used. Accordingly, the present disclosure relates to an in vitro immunoassay comprising a binding molecule, e.g., an antibody of the invention or a DPR-binding fragment thereof.

In another embodiment of the invention, DPR binding molecules, in particular antibodies of the invention, may also be used in a method of diagnosing a disease or disorder in an individual by obtaining a biopsy from the individual tested. In this respect, the invention also relates to a device specifically designed for this purpose. For example, antibody-based arrays may be used, e.g. loaded with an antibody of the invention that specifically recognizes DPR or an equivalent DPR-binding molecule. The design of microarray immunoassays is outlined in Kusnezow et al, mol. cell Proteomics 5(2006), 1681-1696. Thus, the invention also relates to a microarray loaded with a DPR binding molecule of the invention.

In one embodiment, the invention relates to a method of diagnosing a disease or disorder associated with DPR (in particular an aggregated species of DPR, e.g. C9orf72-DPR) in a subject, the method comprising determining the presence of DPR and aggregated DPR, respectively, in a sample from a subject to be diagnosed as having at least one antibody of the invention, a DPR binding fragment thereof, or a DPR binding molecule having substantially the same binding specificity for any of them, wherein the presence of DPR or a pathologically aggregated form thereof, optionally C9orf72-DPR, is indicative for FTLD and/or ALS, and an increased level of DPR or a pathologically aggregated form thereof (in particular C9orf72-DPR) compared to the level of physiological C9orf72 (i.e. not showing translation of repeated sequence regions into DPR protein) is indicative for the progression of FTLD and/or ALS in said subject.

The subject to be diagnosed may be asymptomatic or preclinical for the disease. Optionally, the control subject has a disease associated with DPR, aggregated DPR, and/or optionally C9orf72-DPR, e.g., FTLD, ALS, and FTLD-ALS as described above, among others, wherein similarity between DPR (e.g., aggregated C9orf72-DPR) and the level of a reference standard indicates that the subject to be diagnosed has, or is at risk of developing, a disease and/or disorder associated with DPR aggregation. Alternatively or additionally, as a second control, the control subject does not have DPR aggregation, wherein a difference between the level of physiological C9orf72 or another protein susceptible to insertion into DPR due to a mutation in its gene (such as a mutated C9orf72 gene and/or aggregated C9orf72-DPR) and a reference standard indicates that the subject to be diagnosed has, or is at risk of developing, a disease and/or disorder associated with DPR, such as FTLD, ALS, and/or FTLD-ALS. Optionally, the subject to be diagnosed and the one or more control subjects are age-matched. The sample to be analyzed may be any body fluid suspected of containing pathological DPR proteins, such as aggregated C9orf72-DPR, for example blood, plasma, serum, urine, peritoneal fluid, saliva or cerebrospinal fluid (CSF).

The level of physiological C9orf72 or similar proteins and/or aggregated DPRs, such as C9orf72-DPRs, can be assessed by any suitable method known in the art, including, for example, analyzing DPRs and/or DPR-incorporating proteins, such as C9orf72, by one or more techniques selected from the group consisting of: western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), Fluorescence Activated Cell Sorting (FACS), two-dimensional gel electrophoresis, Mass Spectrometry (MS), matrix-assisted laser desorption/ionization time-of-flight-MS (MALDI-TOF), surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF), High Performance Liquid Chromatography (HPLC), Fast Protein Liquid Chromatography (FPLC), multidimensional Liquid Chromatography (LC) after tandem mass spectrometry (MS/MS), and laser densitometry. Optionally, the in vivo imaging of DPR comprises scintigraphy, Positron Emission Tomography (PET), single photon emission tomography (SPECT), Near Infrared (NIR) optical imaging, or Magnetic Resonance Imaging (MRI).

Thus, in one embodiment, an antibody, polynucleotide, vector or cell as defined above or a pharmaceutical or diagnostic composition comprising any of them of the invention is provided for use in the prophylactic treatment, therapeutic treatment and/or monitoring the progression of or response to treatment of a disease or disorder associated with a DPR protein or aggregated form thereof. Accordingly, the invention also relates to a method of diagnosing or monitoring the progression of a disease or disorder associated with a DPR protein (e.g. FTLD and ALS) in a subject, the method comprising determining the presence of a DPR protein in a sample from a subject to be diagnosed as having at least one antibody of the invention or a DPR binding molecule having substantially the same binding specificity of either, wherein the presence of DPR (e.g. in mutant C9orf72 and aggregated C9orf72-DPR species) is indicative of the disease or disorder. In one embodiment, there is provided the method of diagnosing or monitoring progression of a DPR-associated disease and/or disorder in a subject, the method comprising determining the presence of DPR (such as in mutated C9orf72 and aggregated forms thereof) in a sample from a subject to be diagnosed with at least one antibody of the invention, wherein the presence of DPR (such as mutated C9orf72 and/or aggregated C9orf72-DPR) is indicative of a pre-symptomatic, prodromal or clinical disease and/or disorder associated with DPR, an increase in the level of DPR aggregates (particularly C9orf72-DPR) compared to the level of physiological C9orf72 without DPR or compared to a reference sample derived from a healthy control subject or a control sample from the same subject is indicative of a pre-symptomatic, prodromal or clinical disease and/or disorder associated with DPR, such as FTLD and ALS. The skilled person will understand that in one embodiment the method is also used for diagnosing or monitoring the progression of any other disease or disorder from the group of disorders related to DPR and proteins containing DPR, respectively, as defined above.

As mentioned above, the antibodies of the invention can be used not only in vitro, but also in vivo, where therapeutic applications are sought in addition to diagnostics. Thus, in one embodiment, the invention also relates to a DPR binding molecule comprising the CDRs of an antibody of the invention for use in the preparation of a composition for in vivo detection of DPR (optionally C9orf72-DPR) or a therapeutic and/or diagnostic agent targeted against DPR (optionally C9orf72-DPR) in the human or animal body. The potential therapeutic and/or diagnostic agents may be selected from a non-exhaustive list of therapeutic agents that may be used to treat diseases and/or disorders associated with DPR and the potential markers described above. With respect to in vivo imaging, in a preferred embodiment, the invention provides said DPR binding molecule comprising a CDR of an antibody of the invention, wherein said in vivo imaging comprises scintigraphy, Positron Emission Tomography (PET), single photon emission tomography (SPECT), Near Infrared (NIR) optical imaging or Magnetic Resonance Imaging (MRI). In another embodiment, the invention also provides said DPR binding molecule comprising the CDRs of an antibody of the invention, or said molecule for use in the preparation of a composition for use in the above-specified in vivo imaging method, in a method for diagnosing or monitoring the progression of a disease or disorder associated with a DPR protein in a subject as defined above.

In this case, the invention also relates to a kit useful for diagnosing or monitoring the progression of diseases and/or disorders associated with DPR and proteins comprising DPR, said kit comprising at least one antibody of the invention or a DPR binding molecule having substantially the same binding specificity for any of them, a polynucleotide, vector or cell and/or peptide as defined above respectively, optionally with reagents and/or instructions for use.

Provided herein are compositions, methods and/or uses described by the following numbered paragraphs:

1. an anti-DPR antibody or fragment thereof comprising

(i) A heavy chain having the amino acid sequence of SEQ ID NO: 38 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38); and

(ii) a light chain having the sequence of SEQ ID NO: 42 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42).

An anti-DPR antibody or fragment thereof (e.g., binds, e.g., specifically binds and/or binds with high affinity to a poly- (GA) n repeat, e.g., a poly- (GA) n repeat described herein), e.g., a poly- (GA) _6-15) (SEQ ID NO: 83) wherein the anti-DPR antibody or fragment thereof comprises:

(ii) a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 39 (or an amino acid sequence which is at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39), consists of or consists essentially of,

(iii) A heavy chain variable region amino acid sequence comprising SEQ ID NO: 40 (or an amino acid sequence which is at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 40), consists or essentially consists thereof,

(iv) a light chain amino acid sequence comprising SEQ ID NO: 41 or 42 (or an amino acid sequence which is at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41 or 42), consists or essentially consists thereof,

optionally, wherein the light chain amino acid sequence is no more than 214 amino acids in length, and/or wherein the light chain variable region amino acid sequence is no more than 107 amino acids in length;

(v) a light chain constant domain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 43 (or an amino acid sequence at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 43), consists of or essentially consists of,

Optionally, wherein the light chain amino acid sequence is no more than 214 amino acids in length, and/or wherein the light chain variable region amino acid sequence is no more than 107 amino acids in length;

(vi) a light chain variable region amino acid sequence comprising SEQ ID NO: 44 (or an amino acid sequence at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 44), consists or consists essentially of,

(vii) a heavy chain variable region comprising SEQ ID NO: 45 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 45); SEQ ID NO: 46 (or an amino acid sequence at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 46), and the CDR2 amino acid sequence of SEQ ID NO: 47 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47),

and/or

(viii) A light chain variable region comprising SEQ ID NO: 48 (or an amino acid sequence at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 48); SEQ ID NO: 49 (or an amino acid sequence at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49), and the CDR2 amino acid sequence of SEQ ID NO: 50 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 50),

optionally, wherein the heavy chain amino acid sequence (e.g., heavy chain mature sequence) is no more than 452 amino acids in length, and/or wherein the heavy chain does not comprise a lysine residue at the C-terminus of its amino acid sequence, and/or wherein the heavy chain has a glycine at the C-terminus of its amino acid sequence, and/or wherein the heavy chain variable region amino acid sequence is no more than 123 amino acids in length; and/or optionally, wherein the light chain amino acid sequence is no more than 214 amino acids in length, and/or wherein the light chain variable region amino acid sequence is no more than 107 amino acids in length.

The anti-DPR antibody or fragment thereof of any of paragraphs 1-1e, comprising a heterologous sequence, e.g., that is heterologous to the heavy chain, light chain, heavy chain variable region, heavy chain constant domain, light chain variable region, light chain constant domain, variable heavy CDR, and/or variable light CDR.

The anti-DPR antibody or fragment thereof of paragraph 1b, wherein the heterologous sequence comprises an immunoglobulin heavy chain constant region, an immunoglobulin light chain constant region, or a heterologous mammalian secretory signal peptide.

An anti-DPR antibody or fragment thereof as described in any of paragraphs 1-1c, comprising a polyethylene glycol or a detectable label, e.g., an enzyme, a radioisotope, a fluorescent compound, a chemiluminescent compound, a bioluminescent compound or a heavy metal.

An anti-DPR antibody or fragment thereof as recited in any of paragraphs 1-1d, wherein the antibody or fragment thereof is selected from the group consisting of: fab, Fab′、F(ab′)₂Fc, Fv, single chain Fv (scFv), single chain antibody, and disulfide-linked Fv (sdFv).

2. A nucleic acid molecule comprising:

(i) a nucleic acid sequence encoding a heavy chain of an anti-DPR antibody, said heavy chain having the amino acid sequence of SEQ ID NO: 38 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38); and/or

(ii) A nucleic acid sequence encoding a light chain of an anti-DPR antibody, the light chain having the amino acid sequence of SEQ ID NO: 42 (or an amino acid sequence at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 42), optionally wherein the nucleic acid sequences (i) and (ii) are located on the same nucleic acid molecule or on separate nucleic acid molecules.

The nucleic acid molecule of paragraph 2, wherein the nucleic acid molecule comprises a cDNA and/or is operably linked to a heterologous nucleic acid, such as a heterologous signal peptide (e.g., a secretion signal peptide, e.g., a mammalian secretion signal peptide, e.g., a secretion signal peptide described herein) or a heterologous regulatory element (e.g., a heterologous enhancer, ribosome binding site, transcription terminator, or a heterologous promoter (e.g., a cytomegalovirus, simian virus 40, or retroviral promoter)).

A nucleic acid molecule encoding one or more of the following (i) - (viii):

(iii) a heavy chain variable region amino acid sequence comprising SEQ ID NO: 40 (or an amino acid sequence which is at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 40), consists or essentially consists thereof,

(vi) A light chain variable region amino acid sequence comprising SEQ ID NO: 44 (or an amino acid sequence at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 44), consists or consists essentially of,

And/or

optionally, wherein the heavy chain amino acid sequence (e.g., heavy chain mature sequence) is no more than 452 amino acids in length, and/or wherein the heavy chain does not comprise a lysine residue at the C-terminus of its amino acid sequence, and/or wherein the heavy chain has a glycine at the C-terminus of its amino acid sequence, and/or wherein the heavy chain variable region amino acid sequence is no more than 123 amino acids in length; and/or optionally, wherein the light chain amino acid sequence is no more than 214 amino acids in length, and/or wherein the light chain variable region amino acid sequence is no more than 107 amino acids in length.

3. A nucleic acid molecule comprising SEQ ID NO: 51-58 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 51-58).

One or more nucleic acid molecules encoding the antibody or fragment thereof of any one of paragraphs 1-1 e.

A cDNA comprising the nucleic acid molecule of any of paragraphs 2-3 a.

A cDNA comprising a polynucleotide encoding:

(ii) A heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 39 (or an amino acid sequence which is at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39), consists of or consists essentially of,

(iv) A light chain amino acid sequence comprising SEQ ID NO: 41 or 42 (or an amino acid sequence which is at least 95%, e.g. 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41 or 42), consists or essentially consists thereof,

And/or

optionally, wherein the heavy chain amino acid sequence (e.g., heavy chain mature sequence) is no more than 452 amino acids in length, and/or wherein the heavy chain does not comprise a lysine residue at the C-terminus of its amino acid sequence, and/or wherein the heavy chain has a glycine at the C-terminus of its amino acid sequence, and/or wherein the heavy chain variable region amino acid sequence is no more than 123 amino acids in length; and/or optionally, wherein the light chain amino acid sequence is no more than 214 amino acids in length, and/or wherein the light chain variable region amino acid sequence is no more than 107 amino acids in length; and/or

(ix) A heavy chain having the amino acid sequence of SEQ ID NO: 38 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38); and/or

(x) A light chain having the sequence of SEQ ID NO: 42 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42).

4. A vector comprising the nucleic acid molecule of any one of paragraphs 2-3a or the cDNA of any one of paragraphs 3b-3 c.

The vector of paragraph 4, wherein the vector is an expression vector operably linked to a polynucleotide, wherein the polynucleotide encodes one or more of:

And/or

optionally, wherein the heavy chain amino acid sequence (e.g., heavy chain mature sequence) is no more than 452 amino acids in length, and/or wherein the heavy chain does not comprise a lysine residue at the C-terminus of its amino acid sequence, and/or wherein the heavy chain has a glycine at the C-terminus of its amino acid sequence, and/or wherein the heavy chain variable region amino acid sequence is no more than 123 amino acids in length; and/or optionally, wherein the light chain amino acid sequence is no more than 214 amino acids in length, and/or wherein the light chain variable region amino acid sequence is no more than 107 amino acids in length; and/or

(ix) A heavy chain having the amino acid sequence of SEQ ID NO: 38 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38); and/or

(x) A light chain having the sequence of SEQ ID NO: 42 (or an amino acid sequence that is at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42).

The vector of any one of paragraphs 4-4a, wherein the vector comprises the cDNA of any one of paragraphs 3b-3 c.

The vector of any one of paragraphs 4-4b, wherein the vector comprises a promoter (e.g., a heterologous promoter, such as a cytomegalovirus (e.g., cytomegalovirus immediate early promoter), simian virus 40, or a retroviral promoter).

5. A host cell comprising (i) the nucleic acid molecule of any one of paragraphs 2-3 a; (ii) a cDNA as described in any of paragraphs 3b-3 c; or (iii) a vector as described in any of paragraphs 4-4c,

optionally, wherein the host cell is a mammalian host cell (e.g., a Chinese Hamster Ovary (CHO) cell, a HEK 293 cell, or a NS0 cell).

6. Use of a nucleic acid molecule according to any of paragraphs 2-3a, a cDNA according to any of paragraphs 3b-3c, a vector according to any of paragraphs 4-4c or a host cell according to paragraph 5 for the production of an anti-DPR antibody or a fragment thereof.

7. A method of producing an anti-DPR antibody or fragment thereof, the method comprising: (i) culturing the host cell of paragraph 5; and (ii) isolating the antibody or fragment thereof from the culture.

8. A composition, e.g., a pharmaceutical composition, comprising an anti-DPR antibody or fragment thereof according to any of paragraphs 1-1e, a nucleic acid molecule according to any of paragraphs 2-3a, a cDNA according to any of paragraphs 3b-3c, a vector according to any of paragraphs 4-4c, or a host cell according to paragraph 5,

optionally, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier,

optionally, wherein the pharmaceutical composition is suitable for intrathecal administration.

9. A method of treating a disorder associated with or caused by a DPR-containing protein or an aggregated form thereof (e.g., Amyotrophic Lateral Sclerosis (ALS), frontotemporal lobar degeneration (FTLD), or FTLD-ALS) in a subject in need thereof, the method comprising administering to the subject an anti-DPR antibody or fragment thereof described herein (e.g., an anti-DPR antibody or fragment thereof as described in any of paragraphs 1-1 e), thereby treating the disorder in the subject (e.g., ALS, FTLD, or FTLD-ALS).

10. A method of making a pharmaceutical composition for treating a disorder associated with or caused by a DPR-containing protein or aggregated form thereof (e.g., ALS, FTLD, or FTLD-ALS), the method comprising: (i) culturing the host cell of paragraph 5; (ii) isolating and/or purifying the antibody or fragment thereof from the culture that reaches pharmaceutical grade; and (iii) mixing the antibody or fragment thereof with a pharmaceutically acceptable carrier.

11. An anti-DPR antibody or fragment thereof according to any of paragraphs 1-1e, a nucleic acid molecule according to any of paragraphs 2-3a, a cDNA according to any of paragraphs 3b-3c, a vector according to any of paragraphs 4-4c, or a host cell according to paragraph 5, for use in the treatment (e.g. prophylactic and/or therapeutic treatment) of a disorder associated with or caused by a DPR-containing protein or aggregated form thereof (e.g. ALS, FTLD or FTLD-ALS).

12. A method for detecting DPR (e.g., poly-GA DPR) deposits in the brain in vivo, the method comprising:

administering to a subject (e.g., a human subject) an anti-DPR antibody or fragment thereof as described in any of paragraphs 1-1e, wherein the antibody or fragment thereof is linked to a detectable label (e.g., an enzyme, a radioisotope, a fluorophore, and/or a heavy metal); and

Detecting the detectable label in the brain of the subject, thereby detecting the DPR deposits in the brain of the subject, optionally wherein the DPR deposits are monitored by Positron Emission Tomography (PET), single photon emission tomography (SPECT), Near Infrared (NIR) optical imaging, or Magnetic Resonance Imaging (MRI).

Sequence of

TABLE 14 selection of sequences

The above disclosure generally describes the present application. Unless otherwise indicated, terms as used herein are defined as provided in Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised version 2000 and reissue 2003, ISBN 0198506732. Full bibliographic citations may be found at the end of the specification before the claims. The contents of all cited references (including literature references, issued patents, published patent applications cited throughout this application, including the background section and manufacturer's specifications, descriptions, etc.) are hereby expressly incorporated by reference; however, there is no admission that any of the documents cited are indeed prior art with respect to the present invention.

A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.

Examples

Example 1: isolation and characterization of anti- (poly-GA) dipeptide repeat (DPR) protein antibodies

Human antibodies targeting poly-GA dipeptide repeat (DPR) protein, fragments thereof, C9orf72-DPR and/or fragments thereof were identified based on the methods described in international application WO 2016/050822 a2, the disclosure of which is incorporated herein by reference. In particular, the poly-GA dipeptide repeat protein GA was synthesized and purified by Schafer-N (Copenhagen, Denmark)₁₅：H-CHHHHHH(GA)₁₅-OH) (SEQ ID NO: 61). The poly-GA dipeptide repeat protein was then conjugated to Bovine Serum Albumin (BSA) via a bifunctional linker (SMCC). Subsequently, direct ELISA was performed directly using 96-well microplate (Corning) coated with coating buffer (15mM Na)₂CO₃，35mM NaHCO₃pH 9.42) at a concentration of 5. mu.g/ml of unconjugated or BSA conjugated poly-GA dipeptide repeat protein or BSA (Sigma-Aldrich, Buchs, Switzerland). At room temperature with PBS/0.1% containing 2% BSA (Sigma-Aldrich, Buchs, Switzerland)20 blocking of non-specific binding sites for 1 hour. B cell conditioned media was transferred from memory B cell culture plates to ELISA plates and incubated for 1 hour at room temperature, then incubated with an donkey anti-human IgG Fc γ -specific antibody conjugated to HRP (Jackson ImmunoResearch Laboratories, inc., West Grove, USA) and a goat anti-human IgA-specific antibody conjugated to HRP (Jackson ImmunoResearch Laboratories, inc., West Grove, USA). Binding was determined by measuring HRP activity in a standard colorimetric assay. Antibody cloning was performed only on B cell cultures showing that the antibodies contained in the medium bound to poly-GA DPR, but not to BSA.

Example 2: determination of antibody sequences

The amino acid sequences of the variable regions of the anti- (poly-GA) DPR antibodies identified above were determined based on their mRNA sequences, see fig. 1A-F. Briefly, viable B cells of selected non-immortalized memory B cell cultures are harvested. Subsequently, mRNA was extracted from the cells producing the selected anti- (poly-GA) DPR antibody and converted into cDNA, and the sequence encoding the variable region of the antibody was amplified by PCR, cloned into a plasmid vector and sequenced. Briefly, primer combinations representing all sequence families of the human immunoglobulin germline repertoire were used for amplification of the leader peptide, V-segment, and J-segment. A first round of amplification was performed using leader peptide-specific primers at the 5 'end and constant region-specific primers at the 3' end (Smith et al, Nat Protoc.4(2009), 372-384). For the heavy and kappa light chains, a second round of amplification was performed using V-segment specific primers at the 5 'end and J-segment specific primers at the 3' end. For lambda light chains, a second round of amplification was performed using V-segment specific primers at the 5 'end and C-region specific primers at the 3' end (Marks et al, mol.biol.222(1991), 581-182597; de Haard et al, J.biol.chem.26(1999), 18218-18230).

After recombinant expression of the intact antibody, identification of antibody clones with the desired specificity was performed by rescreening on ELISA. Recombinant expression of the full human IgG1 antibody was achieved by inserting the variable heavy and light chain sequences "in the correct reading frame" into an expression vector that was supplemented at the 5 'end with sequences encoding the leader peptide and at the 3' end with sequences encoding the appropriate constant domains. For this purpose, the primers contain restriction sites designed to facilitate cloning of the variable heavy and light chain sequences into the antibody expression vector. Heavy chain immunoglobulins are expressed by in-frame insertion of immunoglobulin heavy chain RT-PCR products into a heavy chain expression vector with a signal peptide and the constant domain of human or mouse immunoglobulin gamma 1. Kappa light chain immunoglobulins were expressed by in-frame insertion of the kappa light chain RT-PCR product into a light chain expression vector providing the signal peptide and constant domains of human kappa light chain immunoglobulins. Lambda light chain immunoglobulins are expressed by in-frame insertion of a lambda light chain RT-PCR product into a lambda light chain expression vector that provides a signal peptide and constant domains of human or mouse lambda light chain immunoglobulins.

Functional recombinant monoclonal antibodies were obtained when co-transfected into HEK 293 or CHO cells (or any other appropriate human or mouse derived receptor cell line) of an Ig heavy chain expression vector and a kappa or lambda Ig light chain expression vector. The recombinant human monoclonal antibody is then purified from the conditioned medium using standard protein a column purification. An unlimited number of recombinant human monoclonal antibodies can be produced using transiently or stably transfected cells. Cell lines producing recombinant human monoclonal antibodies can be established by direct use of the Ig expression vector or by recloning the Ig variable regions into different expression vectors. Derivatives such as F (ab), F (ab)2 and scFv can also be generated from these Ig variable regions.

The framework regions and complementarity determining regions were determined by comparison with reference antibody sequences available in databases such as Abysis (http:// www.bioinf.org.uk/Abysis /), and annotated using the Kabat numbering scheme (http:// www.bioinf.org.uk/abs /).

Example 3: ELISA EC against C9orf72 dipeptide repeat protein₅₀Analysis of

To determine the binding specificity and half maximal Effective Concentration (EC) of the recombinant humanized C9orf72 antibody NI-308.5J10 to C9orf72 poly-GA DPR₅₀) ELISA EC was performed₅₀And (6) analyzing. Briefly, dipeptide repeat protein was synthesized and purified by Schafer-N (Copenhagen, Denmark): (GA) ₁₅：H-CHHHHHH(GA)₁₅-OH(SEQ ID NO：61)；(GP)₁₅：H-C(GP)₁₅-OH(SEQ ID NO：62)；(GR)₁₅：H-C(GR)₁₅-OH(SEQ ID NO：63)；(PA)₁₅：H-C(PA)₁₅-OH(SEQ ID NO：64)；(PR)₁₅：H-C(PR)₁₅OH (SEQ ID NO: 65). A96-well microplate (Corning Incorporated, Corning, USA) was coated with a coating buffer (15mM Na)₂CO₃，35mM NaHCO₃pH 9.42) at a concentration of 5. mu.g/ml or 20. mu.g/ml. At room temperature with PBS/0.1% containing 2% BSA (Sigma-Aldrich, Buchs, Switzerland)20 blocking of non-specific binding sites for 1 hour. NI-308.5J10 was diluted to the indicated concentration and incubated at room temperature for 1 hour, then incubated with an HRP conjugated donkey anti-human IgG Fc γ specific antibody (Jackson ImmunoResearch Laboratories, inc., West Grove, USA). Binding was determined by measuring HRP activity in a standard colorimetric assay.

EC was estimated by non-linear regression using GraphPad Prism software (San Diego, USA)₅₀The value is obtained. Human antibody NI-308.5J10 to C9orf72 dipeptide repeat protein peptide (GA)₁₅(SEQ ID NO：66)、(GP)₁₅(SEQ ID NO：67)、(GR)₁₅(SEQ ID NO：68)、(PA)₁₅(SEQ ID NO: 69) and (PR)₁₅(SEQ ID NO: 70) antibody NI-308.5J10 specifically recognized poly-GA DPR protein with a binding affinity in the sub-nanomolar range (Table 3, FIG. 2). In general, by RTM^TMScreening high throughput immune repertoire analysis on a healthy elderly donor population resulted in successful cloning and recombinant production of human monoclonal antibodies specifically targeting C9orf72 hexanucleotide amplification-related poly-GA DPRs with high affinity.

Table 3: human antibody NI-308.5J10 for five C9orf72 DPR eggsWhite EC₅₀And (6) analyzing.

Example 4: binding affinity to BSA coupled DPR peptides

To determine the half maximal Effective Concentration (EC) of recombinant humanized NI-308.5J10 antibody on poly-GA C9orf72 dipeptide repeat protein peptide conjugated to Bovine Serum Albumin (BSA)₅₀) ELISA EC was performed₅₀And (6) analyzing. Briefly, poly-GA dipeptide repeat protein was synthesized and purified by Schafer-N (Copenhagen, Denmark): (GA)₁₅：H-CHHHHHH(GA)₁₅OH (SEQ ID NO: 61). The poly-GA DPR protein peptide was then coupled to Bovine Serum Albumin (BSA) via a bifunctional linker (SMCC). A96-well microplate (Corning Incorporated, Corning, USA) was coated with a coating buffer (15mM Na)₂CO₃，35mM NaHCO₃pH 9.42) at a concentration of 5. mu.g/ml of poly-GA BSA coupled or uncoupled dipeptide repeat protein peptide. At room temperature with PBS/0.1% containing 2% BSA (Sigma-Aldrich, Buchs, Switzerland)20 blocking of non-specific binding sites for 1 hour. NI-308.5J10 was diluted to the indicated concentration and incubated at room temperature for 1 hour, then incubated with an HRP conjugated donkey anti-human IgG Fc γ specific antibody (Jackson ImmunoResearch Laboratories, inc., West Grove, USA). Binding was determined by measuring HRP activity in a standard colorimetric assay.

EC was estimated by non-linear regression using GraphPad Prism software (San Diego, USA)₅₀The value is obtained. The comparable binding affinities of BSA-coupled and unconjugated poly-GA DPRs to antibody NI-308.5J10 were determined (table 4, fig. 3). In summary, under these experimental conditions, antibody NI-308.5J10 recognized poly-GA DPR peptide coupled to BSA carrier protein, comparable to affinity to the hydrophobically coated peptide.

Table 4: binding affinity to BSA-coupled and unconjugated C9orf72 DPR peptides.

Example 5: analysis of binding specificity for unrelated amyloidogenic proteins

To determine the target specificity of the NI-308.5J10 recombinant antibody, an indirect ELISA was performed as follows. A96-well microplate (Corning Incorporated, Corning, USA) was coated with a coating buffer (15mM Na)₂CO₃，35mM NaHCO₃pH 9.42) at a concentration of 5. mu.g/ml per peptide (GA)₁₅(SEQ ID NO：66)、(GP)₁₅(SEQ ID NO：67)、(GR)₁₅(SEQ ID NO：68)、(PA)₁₅(SEQ ID NO: 69) or (PR)₁₅(SEQ ID NO: 70) peptide or an unrelated target protein at a concentration of 5-10. mu.g/ml. At room temperature with PBS/0.1% containing 2% BSA (Sigma-Aldrich, Buchs, Switzerland)Non-specific binding sites were blocked for 1 hour. The NI-308.5J10 antibody was diluted to 4nM concentration and incubated at room temperature for 1 hour. Binding was determined using donkey anti-human IgG Fcg specific antibody conjugated to HRP (Jackson ImmunoResearch Laboratories, inc., West Grove, USA) and HRP activity was then measured in a standard colorimetric assay. The signal of the target protein was calculated as fold increase above the median. Target specificity of the NI-308.5J10 human antibody was determined by assessing the binding of the antibody to the C9orf72 dipeptide repeat protein and 7 unrelated amyloid-forming proteins (IAPP, Ab, HD, TTR, a-syn, Tau, TDP-43) by indirect ELISA. As shown in fig. 4, the human antibody NI-308.5J10 showed high binding specificity for the poly-GADPR peptide with no or minimal cross-reactivity to unrelated amyloidogenic proteins.

Example 6: western blot analysis of C9orf72 dipeptide repeat protein

To determine the binding specificity of the recombinant humanized C9orf72 antibody NI-308.5J10 to C9orf72 poly-GA dipeptide repeat protein, immunoblot analysis was performed. Dipeptide repeat protein peptides were synthesized and purified by Schafer-N (Copenhagen, Denmark): (GA)₁₅：H-CHHHHHH(GA)₁₅-OH(SEQ ID NO：61)；(GP)₁₅：H-C(GP)₁₅-OH(SEQ ID NO：62)；(GR)₁₅：H-C(GR)₁₅-OH(SEQ ID NO：63)；(PA)₁₅：H-C(PA)₁₅-OH(SEQ ID NO：64)；(PR)₁₅：H-C(PR)₁₅OH (SEQ ID NO: 65). The DPR protein peptide was then conjugated to Bovine Serum Albumin (BSA) via a bifunctional linker (SMCC). In brief, use of antioxidants supplementedMES SDS running buffer (Life Technologies Europe B.V., Zug, Switzerland) was passed through gradient SDS-PAGEBis-Tris4% -12%; life Technologies Europe B.V., Zug, Switzerland) resolved BSA conjugated dipeptide repeat protein peptide (0.5. mu.g). Then by usingTransfer buffer 2x (Life Technologies Europe B.V., Zug, Switzerland) resolved Western blot (Semi-Dry Blotter, 1 hour, 25V) on a methanol activated PVDF membrane (C)Transfer Membrane，Merck&Cie, Schaffhausen, Switzerland). PBS/0.1% containing 2% BSA (Sigma-Aldrich, Buchs, Switzerland) at 4%(PBST) blocking of non-specific binding sites overnight (or alternatively at room temperature for 1 hour). NI-308.5J10 antibody was diluted to 10nM concentration and incubated at room temperature for 1 hour (or Alternatively at 4 ℃ overnight). The membrane was washed 3 times in PBST at room temperature for 15 minutes and then incubated with donkey anti-human IgG Fc γ -specific antibody conjugated with HRP (1: 20000 or 1: 10000 dilution, Jackson ImmunoResearch Laboratories, Inc., West Grove, USA) for 1 hour at room temperature. Antibody binding was determined by membrane development using ECL and ImageQuant 350 assays (GE Healthcare, Otelfingen, Switzerland).

Western blot analysis to determine the C9orf72 dipeptide repeat protein (GA) of the humanized antibody NI-308.5J10 conjugated to BSA₁₅(SEQ ID NO：66)、(GP)₁₅(SEQ ID NO：67)、(GR)₁₅(SEQ ID NO：68)、(PA)₁₅(SEQ ID NO: 69) and (PR)₁₅(SEQ ID NO: 70). Antibody NI-308.5J10 specifically recognized the DPR protein poly-GA (fig. 5). In summary, human antibody NI-308.5J10 recognized the BSA-conjugated poly-GA DPR peptide after SDS PAGE and western blot. The observed binding pattern was consistent with the results obtained by ELISA analysis.

Example 7: characterization of repeat length-dependent binding by indirect ELISA

To determine the binding affinity of recombinant humanized antibody 308.5J10 to C9orf72 DRP of different repeat sizes, ELISA EC was performed₅₀And (6) analyzing. Briefly, dipeptide repeat protein peptides were synthesized and purified by Schafer-N (Copenhagen, Denmark): GA ₂₀：H-(GA)₂₀HHHHHH-NH₂(SEQ ID NO：71)；GA₁₀：H-(GA)₁₀HHHHHH-NH₂(SEQ ID NO：72)；GA₆：H-(GA)₆HHHHHH-NH₂(SEQ ID NO：73)；GA₅：H-(GA)₅HHHHHH-NH₂(SEQ ID NO：74)；GA₄：H-(GA)₄HHHHHH-NH₂(SEQ ID NO：75)；GA₃：H-(GA)₃HHHHHH-NH₂(SEQ ID NO：76)；GA₂：H-(GA)₂HHHHHH-NH₂(SEQ ID NO: 77). A96-well microplate (Corning Incorporated, Coming, USA) was coated with a coating buffer (15mM Na)₂CO₃，35mMNaHCO₃pH 9.42) of 50. mu.g/ml dipeptide repeatsThe columin peptide is coated. At room temperature with PBS/0.1% containing 2% BSA (Sigma-Aldrich, Buchs, Switzerland)Non-specific binding sites were blocked for 1 hour. NI-308.5J10 was diluted to the indicated concentration and incubated at room temperature for 1 hour, then incubated with an HRP conjugated donkey anti-human IgG Fc γ specific antibody (Jackson ImmunoResearch Laboratories, inc., West Grove, USA). Binding was determined by measuring HRP activity in a standard colorimetric assay. EC was estimated by non-linear regression using GraphPad Prism software (San Diego, USA)₅₀The value is obtained.

The binding affinity of antibody NI-308.5J10 to C9orf72 poly-GA DPR proteins with different repeat lengths was determined by indirect ELISA after hydrophobic peptide coating. Antibody NI-308.5J10 required at least 6 GA repeats for the first detectable binding. High affinity binding of poly-GADPR with 10 (SEQ ID NO: 79) or 20 (GA) -repeats (SEQ ID NO: 82) was detected, reflecting EC in the sub-nanomolar range₅₀(Table 5, FIG. 6). In summary, the human NI-308.5J10 antibody showed repeat length dependent binding to poly-GADPR, lack of binding to short repeat sizes, and had superior high affinity binding to extended dipeptide repeats.

Table 5: c9orf72 poly-GA repeat length dependent binding of antibody NI-308.5J 10.

Example 8: characterization of binding properties by biolayer interferometry

To determine the NI-308.5J10 antibody vs (GA)₁₅(SEQ ID NO: 66) binding constant (K) of dipeptide repeat (DPR) peptides_D，K_a，K_d) Bio-layer interferometry (BLI) was performed. poly-GA dipeptide repeat protein was synthesized and purified by Schafer-N (Copenhagen, Denmark): (GA)₁₅：H-CHHHHHH(GA)₁₅OH (SEQ ID NO: 61). Freeze-drying (GA) of pure₁₅(SEQ ID NO: 66) was dissolved in DMSO (Sigma-Aldrich, Buchs, Switzerland) at a concentration of 10mg/ml and stored at-20 ℃. Briefly, biolayer interferometry experiments were performed on an Octet RED96 instrument (Pall ForteBio LLC, Fremont, USA). Octet amine-reactive (AR2G) biosensor for (GA)₁₅(SEQ ID NO: 66) covalent immobilization of dipeptide repeat protein peptides. EDC (1-ethyl-3- [ 3-dimethylaminopropyl) solution was applied to AR2G biosensor]A carbodiimide hydrochloride; 20mM in water; pall ForteBio LLC, Fremont, USA) and s-NHS (N-hydroxysulfosuccinimide; 10mM in water; pall ForteBio LLC, Fremont, USA) for 300 seconds, and then the biosensor surface was loaded with 5 μ g/ml (GA) in 10mM acetate buffer (pH 6) (Pall ForteBio LLC, Fremont, USA) ₁₅(SEQ ID NO: 66) peptide for 600 seconds. After peptide loading, the AR2G biosensor was quenched with 1M ethanolamine (pH 8.5) (Pall ForteBio LLC, Fremont, USA) for 300 seconds, washed in kinetic buffer (Pall ForteBio LLC, Fremont, USA) for 120 seconds (baseline), and human NI-308.5J10 antibody association was assessed in diluted kinetic buffer (1: 10 in PBS) at different concentrations (30, 15, 7.5, 3.75, and 1.875nM) for 600 seconds. Antibody dissociation was evaluated in kinetic buffer for 800 seconds. All binding data were referenced by collecting data using PBS-only references. By using Octet system software (Pall ForteBio LLC, Fremont, USA) with simultaneous K_a/K_dGlobal fit and 1: 1 interaction model for data analysis. After fitting, BLI sensorgrams were plotted using Prism software from GraphPad (San Diego, USA).

The NI-308.5J10 antibody binds with high affinity KD (0.15 + -0.02 nM) to poly-GA DPR peptide with high association rate constant (K)_a＝(1.63±0.05)x 10⁵M^-1s^-1) And a dissociation constant (K) in about the same range_d＝2.4±0.4)x 10^-5s^-1) (FIG. 7 and Table 6). In conclusion, antibody NI-308.5J10 recognizes poly-GA DPR peptide with high affinity.

Antibodies	KD(M)	K_a(Ms^-1)	K_d(s^-1)
				NI-308.5J10	(1.5±0.2)x 10^-10	(1.63±0.05)x 10⁵	(2.4±0.4)x 10^-5

Table 6: binding constant (K) of antibody NI-308.5J10 to poly-GA DPR peptide_D、K_a、K_d)

Example 9: determination of competitive binding by biolayer interferometry

To determine the epitope competition group for antibodies NI-308.5J10 and NI-mAb, reference biolayer interferometry (BLI) was performed. poly-GA dipeptide repeat protein peptides were synthesized and purified by Schafer-N (Copenhagen, Denmark): (GA)₁₅：H-CHHHHHH(GA)₁₅OH (SEQ ID NO: 61). Freeze-drying (GA) of pure₁₅(SEQ ID NO: 66) was dissolved in DMSO (Sigma-Aldrich, Buchs, Switzerland) at a concentration of 10mg/ml and stored at-20 ℃. Briefly, biolayer interferometry experiments were performed on an Octet RED96 instrument (Pall ForteBio LLC, Fremont, USA). Octet amine-reactive (AR2G) biosensor for (GA)₁₅(SEQ ID NO: 66) covalent immobilization of dipeptide repeat protein peptides. EDC (1-ethyl-3- [ 3-dimethylaminopropyl) solution was applied to AR2G biosensor]A carbodiimide hydrochloride; 20m in waterM; pall ForteBio LLC, Fremont, USA) and s-NHS (N-hydroxysulfosuccinimide; 10mM in water; pall ForteBio LLC, Fremont, USA) for 300 seconds, and then the biosensor surface was loaded with 5 μ g/ml (GA) in 10mM acetate buffer (pH 6) (Pall ForteBio LLC, Fremont, USA)₁₅(SEQ ID NO: 66) peptide for 600 seconds. After peptide loading, the AR2G biosensor was quenched with 1M ethanolamine (pH 8.5) (Pall ForteBio LLC, Fremont, USA) for 300 seconds and rinsed in kinetic buffer (Pall ForteBio LLC, Fremont, USA) for 120 seconds (baseline). Target binding of NI-308 antibody was then assessed in a pairwise fashion: reference NI-308 antibody (15nM in kinetic buffer (Pall ForteBio LLC, Fremont, USA)) and (GA) ₁₅(SEQ ID NO: 66) peptide binding (duration 800 seconds) was performed directly followed by competitive NI-308 antibody (15nM in kinetic buffer (Pall forteBio LLC, Fremont, USA)) binding (duration 800 seconds). All binding data were referenced by collecting data using PBS-only references. Data analysis was performed by using Octet system software (Pall ForteBio LLC, Fremont, USA). The BLI sensorgrams were plotted using Prism software from GraphPad (San Diego, USA).

Antibody NI-mAb reference and C9orf72 dipeptide repeat protein peptide (GA)₁₅Binding (SEQ ID NO: 66) was abolished by previous binding of the NI-308.5J10 antibody to the target (FIG. 8A), indicating that the NI-mAb reference antibody recognizes a binding epitope that is also targeted by the NI-308.5J10 antibody. Antibody NI-308.5J10 with C9orf72 DPR peptide (GA)₁₅Binding (SEQ ID NO: 66) was not blocked by previous binding of the NI-mAb reference antibody to the target (FIG. 8B), indicating that this antibody potentially recognizes additional conformational epitopes on the poly-GA peptide. In summary, antibodies NI-308.5J10 and the NI-mAb reference recognize a common binding epitope, and antibody NI-308.5J10 potentially recognizes an additional conformational epitope on the poly-GA peptide compared to the antibody NI-mAb reference.

Example 10: antibody integrity analysis by SDS PAGE

To assess the purity and integrity of recombinant human NI-308.5J10 antibody, SDS PAGE analysis has been performed. Briefly, human NI-308.5J10 antibody was expressed by transient transfection of CHO-S cells and(ii) a FPLC systemProtein a affinity purification on GE Healthcare Life Sciences). After desalting on a PD-10 column (GE Healthcare Life Sciences), the antibodies were formulated in PBS. By gradient SD S-PAGE under reducing conditions (Bis-Tris4% -12%; life Technologies Europe B.V., Zug, Switzerland) use of antioxidant supplemented (Life Technologies Europe B.V., Zug, Switzerland)MES SDS running buffer 2 and 10. mu.g of purified recombinant human NI-308.5J10 antibody were resolved and then Coomassie blue stained: (SimplyBlue (TM) Safesain, Life Technologies Europe B.V., Zug, Switzerland). As a result, SDS-PAGE analysis under reducing conditions of recombinant human NI-308.5J10 antibody revealed two major bands, corresponding to antibody heavy and light chains of the expected size. No significant contamination or proteolytic degradation products were detected (fig. 9).

Example 11: binding analysis of DPR aggregate pathology in post-mortem human C9orf72-FTLD and non-neurologic control brain tissue

To assess binding of antibody NI-308.5J10 to C9orf72 dipeptide repeat protein in postmortem cerebellar tissue from human C9orf72-FTLD patients and non-neurological controls, a binding assay has been performed. Briefly, formalin-fixed, paraffin-embedded 5 μm sections from the cerebellum of 3 FTLD patients with C9orf72 hexanucleotide repeat amplification and 1 non-neurological control subject (BiOBANC HCB-IDIBAPS, Barcelona, Spain) were pre-treated by treatment in 1mM EDTA buffer (EDTApH 8.3) for antigen recovery and microwave irradiation for 12 minutes (600W). Quenching of endogenous peroxidase Activity by 3% H at room temperature₂O₂Is carried out for 10 minutes. Non-specific binding sites were blocked with PBS/5% serum (horse/goat)/4% BSA for 1 hour at room temperature. After the blocking step, sections were incubated with human NI-308.5J10 antibody at 20nM concentration overnight at 4 ℃. Detection was performed using biotinylated donkey anti-human IgG (H + L) (1: 350dil, Jackson ImmunoResearch Laboratories, Inc., West Grove, USA) or anti-rabbit secondary antibody (1: 250 dilution, Vector Laboratories; Burlingame, USA), and antibody signal was amplified using the Vector stain Elite ABC kit (Vector Laboratories, Burlingame, USA) and detected with diaminobenzidine (DAB, Thermo Scientific, Rockford, USA). Use of Slides were fixed with a fixing medium (O.KindlerGmbH; Freiburg, Germany). Bright field imaging was performed using a dotsilide VS120 slide scanner (Olympus Schweiz AG, Switzerland). Binding of NI-308.5J10 to pathological C9orf72 dipeptide repeat protein was assessed by immunohistochemical analysis of cerebellar sections from selected FTLD patients and non-neurological control subjects. As shown in fig. 10, human NI-308.5J10 antibody revealed prominent neuronal cytoplasmic inclusions, neuronal nuclear inclusions and dystrophic neurites in the granular cell layer of the cerebellum in the tested C9orf72-FTLD cases. In contrast, the non-neurological control cerebellum was negative for the tested antibody (fig. 10). In summary, the human antibody NI-308.5J10 specifically detected C9orf72-FTLD case cerebellar granule cell layer C9orf72 dipeptide repeat protein, while no staining was observed in control cerebellum, indicating high target specificity of the antibody.

Example 12: identification of light chain glycosylation and heavy chain Asn54 deamidation in NI-308.5J10

To identify post-translational modifications, mass spectrometry has been performed. NI-308.5J10hIgG1 was denatured by heating and treated with RapidGest (Waters, Inc) followed by deglycosylation with PNGase F (Prozyme). After treatment, the proteins were denatured in 4M urea and 40mM DTT in 10mM EDTA for 1 hour at 37 ℃. Rapidest was quenched with 0.5% TFA at 37 ℃ for 1 hour and analyzed on an LCT Premier mass spectrometer (Waters, Inc). The separation of light and heavy chains was achieved on a TSKgel Phenyl-5PW column (2.0X 75mm, 10 μm, TOSOH Bioscience). The generated molecular masses were deconvoluted using MaxEnt 1 software (Waters, Inc). Complete mass analysis of reduced NI-308.5J10 showed that the N-glycosylation site of the light chain was almost completely occupied by the hybrid/complex glycan and that the detected heavy chain corresponded to the predicted pyroGLu 23-475.

After this time, a trypsin digestion/mass spectrometry was performed. Briefly, antibody NI-308.5J10 was reduced, alkylated, precipitated and trypsinized. 2M Urea, 0.15M tris-HCl, 2mM CaCl were used₂Trypsin digestion was carried out for 8 hours at room temperature with 7% (w/w) trypsin (Promega) in methylamine at pH 7.6 and 5 mM. To remove the N-glycans, 1.25mU of PNGasF (Prozyme) was added to the mixture after 6 hours of incubation. Urea was added to digest to a final concentration of 4M prior to LC-MS analysis. The digestion mixture was analyzed on an LC-MS system consisting of UPLC and Xevo G2-S QTof mass spectrometer (Waters, Inc). The separation of the digesta was achieved with an Acquity HSS T3C 18 column (2.1 × 150mM, Waters, Inc) with gradient elution (TFA/acetonitrile). LC-MS peptide mapping data were processed using BiopharmaLynx software. The authentication is verified manually. The amount of modification was estimated from the ion count. Analysis revealed that Asn54 of the heavy chain was very susceptible to deamidation. More than 85% of HC Asn54 in this sample was deamidated, with about 85% in the isoAsp form. The results are summarized in table 7. isoAsp at position 54 is also observed in the crystal structure of the NI308.5J10 Fab.

Oxidation site	Deamidation%
		LC N33	2
HC N54	96
		HC N391	1.7
HC N396	0.3

Table 7: N1308.5J10 deamidation of Asn residue

Example 13: design of NI-308.5J10 variant and validation of mutations

An NI-308.5J10 light chain mutation was selected to remove the light chain glycosylation site (N75D). The four NI-308.5J10 heavy chain mutations were selected to remove either the deamidated asparagine (N54S, N54T) or the glycine at position 55 (G55S, G55T). The constructs were designed to express the variants as intact human IgG 1. To allow Fab expression, constructs containing VH and CH1 regions with variants of the C-terminal hexahistidine tag (SEQ ID NO: 84) were designed. Sequences are listed in

In table 8.

Table 8: modified amino acid sequence of NI-308.5J10 antibody

To verify the mutation, mass spectrometry of the NI-308.5J10 antibody variant N54S/N75D was performed. A complete mass analysis of 5J 10N 54S/N75D hIgG1 showed that the major components detected in the reduced glycosylated antibody were the predicted light chain and the heavy chain with N-linked GOF glycans, indicating the success of the N75D mutation. The mass spectrum of the reduced non-deglycosylated NI-308.5J 10N 54S/N75D hIgG1 was deconvoluted. A complete mass analysis of NI-308.5J 10N 54S/N75D hIgG1 showed that the major components detected in the reduced glycosylated antibody were the predicted light chain and the heavy chain with N-linked GOF glycans, indicating the success of the N75D mutation.

Fig. 11 shows the crystal structure of the NI-308.5J10 antibody into which the mutation has been mapped. As can be derived from the crystal structure, the post-translational modifications are remote from the binding site of the antibody.

Example 14: production of variant IgG1 and Fab

Engineered antibody NI-308.5J10 variants consisting of N75D light chain with each heavy chain mutant were transiently transfected into CHO-S cells using a FectoPro transfection reagent and transferred to reduced temperatures after 24 hours. Using the above constructs, proteins were produced in the form of intact human IgG1 and also in the form of Fab. The supernatant was harvested by centrifugation and clarified by passage through a 0.45um filter. The Fab was then purified by affinity chromatography followed by size exclusion chromatography (table 9). To purify the intact IgG1 protein, the clarified medium was loaded onto rProtein a sepharose (GE healthcare). The column was washed with 20mM Na₂HPO₄Washed with 150mM NaCl, pH 7.4, and 25mM NaH₂PO₄The protein was eluted at 100mM NaCl, pH 2.8, using 12.5mM Na diluted from a 0.5M stock₂HPO₄pH 8.6 neutralization. For purification of Fab, the clarified medium was loaded onto Niexcel agarose gel (GE Healthcare), washed with buffer A (25mM tris pH 8, 500mM NaCl, 10mM imidazole), and eluted with buffer A containing 300mM imidazole. The affinity purified protein was purified on a Superdex 20010/300 column in PBS. The purified proteins were analyzed for size and homogeneity by SDS-PAGE. For SDS-PAGE, the samples were placed in 4% -20% Tris-glycine gradient gels from Invitrogen. The non-reduced sample was heated at 95 ℃ for 3 minutes before electrophoresis. The reduced sample was treated with sample buffer containing 100mM DTT and heated at 95 ℃ for 3 minutes before electrophoresis. The results of SDS-PAGE (FIG. 12) showed that all proteins showed the expected size, with no significant aggregates or protein hydrolysates.

Table 9: expression of NI-308.5J10 variant IgG and Fab

Example 15: binding of proteins to poly-GA as determined by SPR

Binding of the variant fabs to synthetic poly-GA was assessed by SPR using a Biacore T200 instrument (GE Healthcare). Using the reagents and protocols provided by the manufacturer, synthetic biotin 8xGA BSA was prepared at 2-4pg/mM from a solution of 5ng/mL in SPR buffer (10mM HEPES, pH 7.2, 150mM NaCl, 3.4mM EDTA, 0.05% BSA, 0.005% surfactant P20)²Captured on biotin Capture chips (GE Healthcare). A series of solutions of increasing concentrations of 1.23, 3.7, 11, 33 and 100nM variant antibody Fab fragments in SPR buffer were injected at 30 μ L/min each onto the biotin 8xGA coated sensor chip for 4 minutes, followed by buffer washing and binding reactions recorded during injection and within 15 minutes after final injection against the reference sensor without biotin 8 xGA. Data were analyzed using Biacore T200 evaluation software v3.0 using the 1: 1 binding model. All NI-308.5J10 variants showed similar binding kinetics to synthetic 8XGA, where K is _D20-30 nM. The results of the measurement are shown in Table 10. As shown by the SPR binding profile of the NI-308.5J10 variant Fab with synthetic 8 XGA, all NI-308.5J10 variants showed similar binding kinetics with a KD of 20-30nM to the synthetic 8 XGA.

Table 10: binding rate and affinity of the NI-308.5J10 variant Fab to 8x GA as measured by SPR.

Example 16: stability of 5J10 variants

Additional tests of molecular stability were performed on the NI-308.5J10 variant. The thermal stability curves of all variants generated by differential scanning calorimetry (VP-DSC, MicroCal) were similar, with the melting temperature of the CH2 domain (intact IgG1) being about 72 ℃, the melting temperature of the Fab being in the range 79 ℃ to 81 ℃, and the melting temperature of the CH3 domain > 86 ℃ (table 11).

Molecule	Tm₁ CH2	Tm₂ Fab	Tm₃ CH3
				5J10hIgG1	*	80.2	*
5J10-LC N75D hIgG1	72.9	80.0	87.0
				5J10-LC N75D，HC N54S hIgG1	72.6	80.6	87.2
5J10-LC N75D，HC N54T hIgG1	72.9	79.0	86.7
				5J10-LC N75D，HC G55S hIgG1	72.7	79.7	86.9
5J10-LC N75D，HC G55T hIgG1	72.5	79.8	86.9
				5J10Fab	NA	80.2	NA
5J10-LC N75D Fab	NA	80.2	NA
				5J10-LC N75D，HC N54S Fab	NA	80.7	NA
5J10-LC N75D，HC N54T Fab	NA	79.3	NA
				5J10-LC N75D，HC G55S Fab	NA	80.2	NA
5J10-LC N75D，HC G55T Fab	NA	80.2	NA

Table 11: thermostability of the NI-308.5J10 variant for intact IgG, by DSC at temperature T_ml(characterization of unfolding of the hFc CH2 Domain), T_m2(characterisation of unfolding of Fab (CH1, VH, CL, VL)) and T_m3(characterization of the unfolding of the hFc CH3 domain) three major melting transitions were observed. For 5J10 hIgG1, due to T_m2Overlap and failure to determine T_mlAnd T_m3. For Fab, a melting transition was observed, characterizing the unfolding of Fab (CH1, VH, CL, VL).

Example 17: cell-based model for studying the pathogenic mechanism of the C9orf72 DPR protein

Recent reports in emerging cell culture and animal models provide evidence for the toxicity of abnormal C9orf72 DPR protein. For example, toxicity of cytoplasmic poly-GA in cell culture systems has been reported by May et al (Acta Neurophathol.128 (2014), 485- "503) and Zhang et al (Acta Neuropathol.128(2014), 505-" 524).

To determine whether the spread of DPR pathology can be prevented by treatment with the antibodies of the invention as shown for tau (Yanamanda et al, Ann. Clin. Transl. Neurol.2(2013), 278-. In particular, synthetic DNA sequences were generated to drive the expression of individual DPR proteins with 150 dipeptide repeats in ATG-dependent translation. A random codon strategy was employed to ensure that only selected individual DPR protein sequences were expressed. To drive expression of DPR proteins in neuronal cells such as SH-SY5Y, NSC-34, Neuro-2a, iPSC-derived neurons and primary neurons, synthetic DNA sequences were cloned into expression vectors regulated by neuron-specific Thy 1.2 promoters. For high level expression in various eukaryotic cells (e.g., HEK293T, U-2 OS, HeLa and Cos cells), the synthetic DNA sequences were cloned into expression vectors regulated by CMV promoters. Human iPSC-derived neurons and transdifferentiated neurons (iNeurons) derived from C9orf72 patient fibroblasts represent an additional cell culture model for C9orf72 DPR protein.

These cell models can be used to test the therapeutic utility of the antibodies of the invention. Evaluation and confirmation of the therapeutic effect of the antibodies of the invention can be performed by monitoring cell viability via mitochondrial and/or caspase activity assays, monitoring cytotoxicity via cytolytic and/or membrane leakage assays, and monitoring inhibition of cellular DPR protein diffusion via immunohistochemical assays.

Example 18: validation of therapeutic utility of anti-DPR antibodies in transgenic mouse models of C9orf72 pathology

Immunotherapy approaches developed against proteins that are prone to aggregation and/or misfolding have achieved promising results in preclinical and clinical studies of several neurodegenerative diseases. The therapeutic utility of the subject anti-DPR antibodies was validated in a transgenic mouse model of C9orf72 pathology as described in example 15 of WO 2016/050822 a 2. In addition, C9orf72 BAC transgenic mouse strains (Liu et al, Neuron 90(2016), 521-34 and Jiang et al, Neuron 90(2016), 535-50) have been developed that show pathological hallmarks of C9orf72 disease, including RNA foci and dipeptide repeat proteins from RAN translation and associated cognitive deficits and survival phenotypes, which are useful for confirming the therapeutic utility of anti-DPR antibodies. In addition, a suitable transgenic mouse strain, the so-called C9-500BAC transgenic mouse strain expressing the human C9orf72 gene having about 500 hexanucleotide repeats, is commercially available from Jackson Laboratory, 600Main Street, Bar Harbor, ME USA 04609 as FVB/NJ-Tg (C9orf72)500 Lpwr/J. Hemizygous mice of this strain develop age-dependent paralysis, anxiety-like behavior, decreased survival rates, and extensive neurodegeneration of the brain and spinal cord, with accumulation of sense/antisense RNA foci and aggregation of RAN protein and TDP 43. The C9-500 mouse allows the study of acute, rapidly progressive disease as well as slowly progressive disease. This is therefore a model for studying the increased length-dependent toxicity of C9orf72 repeats in familial amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) and frontotemporal dementia (FTD), and is therefore a preferred mouse model for demonstrating the therapeutic utility of the subject antibodies.

In addition, other animal models have been developed; see, e.g., Simone et al (2018), drosophila models described supra, and non-human animal models described in international application WO 2018/064600. Strategies for converting the in vivo data generated from such animal models into therapies for the corresponding human diseases are also known to those skilled in the art; see, e.g., Pichia neuropathology Communications 4(2016), 1-29, by Picher-Martel et al. In particular, guidance can be gained from the development of adtuzumab, a recombinant humanized antibody that is able to target beta-amyloid (Abeta) in the brain of alzheimer's disease patients and has thus far shown promising results in phase I and II clinical trials. The production of lead antibodies for adalimumab and the study of their in vivo biochemical and immunohistochemical properties and biological activity in a mouse model of alzheimer's disease is described in international application WO 2008/081008, the disclosure of which is incorporated herein by reference. As shown in example 4 of WO 2008/081008, anti-Abeta antibodies are able to improve abnormal cognitive behaviour and confer a reduction in beta-amyloid plaque load in a transgenic mouse model of alzheimer's disease when administered intraperitoneally and weakly at a dose of 3 mg/kg. The dose and treatment regimen used in the mouse model also proved to be effective in clinical trials, as could be demonstrated in clinical trials, where doses between 1 and 10mg/kg, including 3mg/kg, have been studied. Thus, a transgenic mouse model of a disease caused by a pathological protein can be well used to predict the therapeutic utility of a given antibody in human patients.

With respect to the mode of administration and dose of the subject antibodies and variants thereof, based on studies of the therapeutic potential of peripheral antibody therapy of transgenic mice overexpressing the disease-causing human superoxide dismutase 1(SOD1) mutant leading to the development of symptoms of Amyotrophic Lateral Sclerosis (ALS), treatment may appear to be effective following direct brain infusion and also peripheral administration of anti-SOD 1 antibody, particularly when administered at 3 to 30mg/kg doses per intraperitoneal (i.p.) injection per week; see, Maier et al, 2018, Science relative Medicine. Since the poly GA-DPR containing protein is translated from the C9orf72 gene, similar to SOD1 with misfolding and aggregation in the brain of patients with ALS, and may even co-aggregate, it was prudently expected that the subject antibodies and variants thereof would be effective within the same dosage regimen, i.e., intraperitoneal injections weekly at 3 to 30 mg/kg. Thus, in a preferred embodiment, the anti-DPR antibodies and DPR-binding fragments thereof are formulated in a pharmaceutical composition designed to be administered once a week via intraperitoneal injection at a dose of 3 to 30 mg/kg. Thus, also according to the present invention, intraperitoneal injections of the subject antibody at a dose of 3 to 30mg/kg once a week is a preferred administration regimen.

Furthermore, due to evolutionary optimization and affinity maturation within the human immune system, the antibodies of the invention provide valuable therapeutic tools due to their isolation from healthy human subjects and their excellent safety and lack of high probability of immunogenicity. Confirmation of these expected therapeutic effects may be provided by the test methods described in the above publications using human antibodies instead of mouse antibodies.

Sequence listing

<110> Biao root MA

Biological Co.,Ltd.

<120> human anti- (poly-GA) dipeptide repeat (DPR) antibodies

<130> 13751-0309WO1

<140>

<141>

<150> 62/772,809

<151> 2018-11-29

<150> EP 18169888.7

<151> 2018-04-27

<160> 84

<170> PatentIn version 3.5

<210> 1

<211> 372

<212> DNA

<213> Intelligent (Homo sapiens)

<220>

<221> CDS

<222> (1)..(372)

<223 >/Note = "NI-308.5J10 variable heavy chain (VH) sequence"

<400> 1

cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gag 48

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

1 5 10 15

acc ctg tcc ctc act tac act gtc tta ggt ggc tcc gtc agt gat tac 96

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

tac tgg agc tgc atc cgg cag ccc gcc ggg aag gga ctg gag tgg att 144

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

35 40 45

ggg cga aca tat act aac ggg aag acc act tac act tac aac ccc tcc 192

Gly Arg Thr Tyr Thr Asn Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

ctc gag agt cga ctc agt ttg tct ata gac acg tcc atg aac caa ttc 240

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

tcc ctg aag ttg acc tct gtg acg gcc gcg gac acg gcc gtc tat tac 288

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

85 90 95

tgc gcg aga tgg ggg gcg gtg act ggt gac tac tac tac ggt atg gac 336

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

gtc tgg ggc cca ggc acc ctg gtc acc gtc tcc tcg 372

Val Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 2

<211> 124

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 2

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Asn Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

Val Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 3

<211> 5

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 3

Asp Tyr Tyr Trp Ser

1 5

<210> 4

<211> 18

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 4

Arg Thr Tyr Thr Asn Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser Leu

1 5 10 15

Glu Ser

<210> 5

<211> 14

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 5

Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp Val

1 5 10

<210> 6

<211> 336

<212> DNA

<213> Intelligent (Homo sapiens)

<220>

<221> CDS

<222> (1)..(336)

<223 >/Note = "NI-308.5J10 variable light chain (VK) sequence"

<400> 6

gaa att gtg ctg act cag tct cca ctc tcc ctg tcc gtc acc cct gga 48

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

1 5 10 15

gag ccg gcc tcc atc tcc tgc agg tct cct cgg agc ctt cta cat act 96

Glu Pro Ala Ser Ile Ser Cys Arg Ser Pro Arg Ser Leu Leu His Thr

20 25 30

aat gga tat aca tat ttg gac tgg tac cta caa agg cca ggg cag tct 144

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

35 40 45

cca caa ctc ctg atc ttt ttg gct tct aat cgg gcc tcc ggg gtc cct 192

Pro Gln Leu Leu Ile Phe Leu Ala Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

gac agg ttc agt ggc agc gga tca ggc aca aat ttt aca ctg aga atc 240

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

65 70 75 80

agc gga gtg gag gct gac gat gtt gga gtt tat tac tgc atg caa ggt 288

Ser Gly Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

cta caa cct tcg tgg acg ttc ggc cag ggg acc aag gtg gaa atc aaa 336

Leu Gln Pro Ser Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 7

<211> 112

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 7

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Pro Arg Ser Leu Leu His Thr

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Phe Leu Ala Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Gly Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Leu Gln Pro Ser Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 8

<211> 16

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 8

Arg Ser Pro Arg Ser Leu Leu His Thr Asn Gly Tyr Thr Tyr Leu Asp

1 5 10 15

<210> 9

<211> 7

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 9

Leu Ala Ser Asn Arg Ala Ser

1 5

<210> 10

<211> 9

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 10

Met Gln Gly Leu Gln Pro Ser Trp Thr

1 5

<210> 11

<211> 372

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<220>

<221> CDS

<222> (1)..(372)

<223 >/Note = "NI-308.5J10 variable heavy chain (VH) sequence-N54S

Mutation "

<400> 11

cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gag 48

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

1 5 10 15

acc ctg tcc ctc act tac act gtc tta ggt ggc tcc gtc agt gat tac 96

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

tac tgg agc tgc atc cgg cag ccc gcc ggg aag gga ctg gag tgg att 144

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

35 40 45

ggg cga aca tat act agc ggg aag acc act tac act tac aac ccc tcc 192

Gly Arg Thr Tyr Thr Ser Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

ctc gag agt cga ctc agt ttg tct ata gac acg tcc atg aac caa ttc 240

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

tcc ctg aag ttg acc tct gtg acg gcc gcg gac acg gcc gtc tat tac 288

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

85 90 95

tgc gcg aga tgg ggg gcg gtg act ggt gac tac tac tac ggt atg gac 336

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

gtc tgg ggc cca ggc acc ctg gtc acc gtc tcc tcg 372

Val Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 12

<211> 124

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 12

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Ser Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

Val Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 13

<211> 18

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<220>

<221> sources

<223 >/Note = "NI-308.5J10 VH-CDR2 sequence-N54S mutation"

<400> 13

Arg Thr Tyr Thr Ser Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser Leu

1 5 10 15

Glu Ser

<210> 14

<211> 372

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<220>

<221> CDS

<222> (1)..(372)

<223 >/Note = "NI-308.5J10 variable heavy chain (VH) sequence-N54T

Mutation "

<400> 14

cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gag 48

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

1 5 10 15

acc ctg tcc ctc act tac act gtc tta ggt ggc tcc gtc agt gat tac 96

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

tac tgg agc tgc atc cgg cag ccc gcc ggg aag gga ctg gag tgg att 144

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

35 40 45

ggg cga aca tat act acc ggg aag acc act tac act tac aac ccc tcc 192

Gly Arg Thr Tyr Thr Thr Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

ctc gag agt cga ctc agt ttg tct ata gac acg tcc atg aac caa ttc 240

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

tcc ctg aag ttg acc tct gtg acg gcc gcg gac acg gcc gtc tat tac 288

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

85 90 95

tgc gcg aga tgg ggg gcg gtg act ggt gac tac tac tac ggt atg gac 336

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

gtc tgg ggc cca ggc acc ctg gtc acc gtc tcc tcg 372

Val Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 124

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 15

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Thr Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

Val Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 16

<211> 18

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<220>

<221> sources

<223 >/Note = "NI-308.5J10 VH-CDR2 sequence-N54T mutation"

<400> 16

Arg Thr Tyr Thr Thr Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser Leu

1 5 10 15

Glu Ser

<210> 17

<211> 372

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<220>

<221> CDS

<222> (1)..(372)

<223 >/Note = "NI-308.5J10 variable heavy chain (VH) sequence-G55S

Mutation "

<400> 17

cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gag 48

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

1 5 10 15

acc ctg tcc ctc act tac act gtc tta ggt ggc tcc gtc agt gat tac 96

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

tac tgg agc tgc atc cgg cag ccc gcc ggg aag gga ctg gag tgg att 144

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

35 40 45

ggg cga aca tat act aac agc aag acc act tac act tac aac ccc tcc 192

Gly Arg Thr Tyr Thr Asn Ser Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

ctc gag agt cga ctc agt ttg tct ata gac acg tcc atg aac caa ttc 240

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

tcc ctg aag ttg acc tct gtg acg gcc gcg gac acg gcc gtc tat tac 288

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

85 90 95

tgc gcg aga tgg ggg gcg gtg act ggt gac tac tac tac ggt atg gac 336

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

gtc tgg ggc cca ggc acc ctg gtc acc gtc tcc tcg 372

Val Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 124

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 18

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Asn Ser Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

Val Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 18

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<220>

<221> sources

<223 >/Note = "NI-308.5J10 VH-CDR2 sequence-G55S mutation"

<400> 19

Arg Thr Tyr Thr Asn Ser Lys Thr Thr Tyr Thr Tyr Asn Pro Ser Leu

1 5 10 15

Glu Ser

<210> 20

<211> 372

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<220>

<221> CDS

<222> (1)..(372)

<223 >/Note = "NI-308.5J10 variable heavy chain (VH) sequence-G55T

Mutation "

<400> 20

cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gag 48

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

1 5 10 15

acc ctg tcc ctc act tac act gtc tta ggt ggc tcc gtc agt gat tac 96

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

tac tgg agc tgc atc cgg cag ccc gcc ggg aag gga ctg gag tgg att 144

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

35 40 45

ggg cga aca tat act aac acc aag acc act tac act tac aac ccc tcc 192

Gly Arg Thr Tyr Thr Asn Thr Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

ctc gag agt cga ctc agt ttg tct ata gac acg tcc atg aac caa ttc 240

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

tcc ctg aag ttg acc tct gtg acg gcc gcg gac acg gcc gtc tat tac 288

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

85 90 95

tgc gcg aga tgg ggg gcg gtg act ggt gac tac tac tac ggt atg gac 336

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

gtc tgg ggc cca ggc acc ctg gtc acc gtc tcc tcg 372

Val Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 21

<211> 124

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 21

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Asn Thr Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

Val Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 22

<211> 18

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<220>

<221> sources

<223 >/Note = "NI-308.5J10 VH-CDR2 sequence-G55T mutation"

<400> 22

Arg Thr Tyr Thr Asn Thr Lys Thr Thr Tyr Thr Tyr Asn Pro Ser Leu

1 5 10 15

Glu Ser

<210> 23

<211> 336

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<220>

<221> CDS

<222> (1)..(336)

<223 >/Note = "NI-308.5J10 variable light chain (VK) sequence-N75D

Mutation "

<400> 23

gaa att gtg ctg act cag tct cca ctc tcc ctg tcc gtc acc cct gga 48

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

1 5 10 15

gag ccg gcc tcc atc tcc tgc agg tct cct cgg agc ctt cta cat act 96

Glu Pro Ala Ser Ile Ser Cys Arg Ser Pro Arg Ser Leu Leu His Thr

20 25 30

aat gga tat aca tat ttg gac tgg tac cta caa agg cca ggg cag tct 144

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

35 40 45

cca caa ctc ctg atc ttt ttg gct tct aat cgg gcc tcc ggg gtc cct 192

Pro Gln Leu Leu Ile Phe Leu Ala Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

gac agg ttc agt ggc agc gga tca ggc aca gac ttt aca ctg aga atc 240

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

65 70 75 80

agc gga gtg gag gct gac gat gtt gga gtt tat tac tgc atg caa ggt 288

Ser Gly Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

cta caa cct tcg tgg acg ttc ggc cag ggg acc aag gtg gaa atc aaa 336

Leu Gln Pro Ser Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 24

<211> 112

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 24

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Pro Arg Ser Leu Leu His Thr

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Phe Leu Ala Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Gly Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Leu Gln Pro Ser Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 25

<211> 219

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> sources

<223 >/Note = "NI-308.5J10 variable light chain (VK) plasmid

(SDD 152)"

<400> 25

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Pro Arg Ser Leu Leu His Thr

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Phe Leu Ala Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Gly Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Leu Gln Pro Ser Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

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

115 120 125

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

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 26

<211> 453

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> sources

<223 >/Note = "NI-308.5J10-hIgG1 variable heavy chain (VH) plasmid

(SDD 151)"

<400> 26

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Asn Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ser Leu Ser Pro Gly

450

<210> 27

<211> 219

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> sources

<223 >/Note = "NI-308.5J10 variable light chain (VK) plasmid (SDD 177)

-N75D mutation "

<400> 27

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Pro Arg Ser Leu Leu His Thr

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Phe Leu Ala Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Gly Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Leu Gln Pro Ser Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

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

115 120 125

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

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 28

<211> 453

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> sources

<223 >/Note = "NI-308.5J10-hIgG1 variable heavy chain (VH) plasmid

(SDD 173) -N54S mutation "

<400> 28

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Ser Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ser Leu Ser Pro Gly

450

<210> 29

<211> 453

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> sources

<223 >/Note = "NI-308.5J10-hIgG1 variable heavy chain (VH) plasmid

(SDD 174) -N54T mutation "

<400> 29

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Thr Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ser Leu Ser Pro Gly

450

<210> 30

<211> 453

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> sources

<223 >/Note = "NI-308.5J10-hIgG1 variable heavy chain (VH) plasmid

(SDD 175) -G55S mutation "

<400> 30

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Asn Ser Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ser Leu Ser Pro Gly

450

<210> 31

<211> 453

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> sources

<223 >/Note = "NI-308.5J10-hIgG1 variable heavy chain (VH) plasmid

(SDD 176) -G55T mutation "

<400> 31

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Asn Thr Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ser Leu Ser Pro Gly

450

<210> 32

<211> 233

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> sources

<223 >/Note = "NI-308.5J10-Fab-6His variable heavy chain (VH) plasmid

(SDD 178)"

<400> 32

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Asn Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Lys Ser Cys His His His His His His

225 230

<210> 33

<211> 233

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> sources

<223 >/Note = "NI-308.5J10-Fab-6His variable heavy chain (VH) plasmid

(SDD 179) -N54S mutation "

<400> 33

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Ser Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Lys Ser Cys His His His His His His

225 230

<210> 34

<211> 233

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> sources

<223 >/Note = "NI-308.5J10-Fab-6His variable heavy chain (VH) plasmid

(SDD 180) -N54T mutation "

<400> 34

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Thr Gly Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Lys Ser Cys His His His His His His

225 230

<210> 35

<211> 233

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> sources

<223 >/Note = "NI-308.5J10-Fab-6His variable heavy chain (VH) plasmid

(SDD 181) -G55S mutant "

<400> 35

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Asn Ser Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Lys Ser Cys His His His His His His

225 230

<210> 36

<211> 233

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> sources

<223 >/Note = "NI-308.5J10-Fab-6His variable heavy chain (VH) plasmid

(SDD 182) -G55T mutation "

<400> 36

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

1 5 10 15

Thr Leu Ser Leu Thr Tyr Thr Val Leu Gly Gly Ser Val Ser Asp Tyr

20 25 30

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

35 40 45

Gly Arg Thr Tyr Thr Asn Thr Lys Thr Thr Tyr Thr Tyr Asn Pro Ser

50 55 60

Leu Glu Ser Arg Leu Ser Leu Ser Ile Asp Thr Ser Met Asn Gln Phe

65 70 75 80

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

85 90 95

Cys Ala Arg Trp Gly Ala Val Thr Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Lys Ser Cys His His His His His His

225 230

<210> 37

<211> 471

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 37

Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg

1 5 10 15

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

20 25 30

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

35 40 45

Ser Asn His Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Glu Asn Thr Tyr Tyr Ala

65 70 75 80

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

85 90 95

Thr Leu Phe Leu Gln Met Tyr Ser Leu Thr Ala Asp Asp Thr Ala Met

100 105 110

Tyr Phe Cys Ala Arg Gly Gly Arg Arg Gly His Phe Thr Ser Tyr Tyr

115 120 125

Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 38

<211> 452

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 38

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn His

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Tyr Ser Leu Thr Ala Asp Asp Thr Ala Met Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Arg Arg Gly His Phe Thr Ser Tyr Tyr Leu Asp Tyr

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Pro Gly

450

<210> 39

<211> 329

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 39

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 40

<211> 123

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 40

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn His

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Tyr Ser Leu Thr Ala Asp Asp Thr Ala Met Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Arg Arg Gly His Phe Thr Ser Tyr Tyr Leu Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 41

<211> 236

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 41

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

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

35 40 45

Gln Asn Ile Asp Lys Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu His Ser Gly Val

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

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

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 42

<211> 214

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 42

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Ile Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Ile Tyr Tyr Cys Gln Gln Ser Tyr Ser Ser Phe Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 43

<211> 107

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 43

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

1 5 10 15

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

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 44

<211> 107

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 44

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Ile Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Ile Tyr Tyr Cys Gln Gln Ser Tyr Ser Ser Phe Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 45

<211> 10

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<400> 45

Gly Phe Thr Phe Ser Asn His Ala Met His

1 5 10

<210> 46

<211> 17

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<400> 46

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

1 5 10 15

Gly

<210> 47

<211> 14

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<400> 47

Gly Gly Arg Arg Gly His Phe Thr Ser Tyr Tyr Leu Asp Tyr

1 5 10

<210> 48

<211> 11

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<400> 48

Arg Ala Ser Gln Asn Ile Asp Lys Tyr Leu Asn

1 5 10

<210> 49

<211> 7

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<400> 49

Ala Ala Ser Ser Leu His Ser

1 5

<210> 50

<211> 9

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<400> 50

Gln Gln Ser Tyr Ser Ser Phe Arg Thr

1 5

<210> 51

<211> 1416

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 51

atgggttgga gcctcatctt gctgtttctt gtcgctgttg ctacgcgtgt cctgtcgcag 60

gtgcagctgg tggagtctgg gggaggcgta gtccagcctg ggaggtccct gagactgtcc 120

tgtgcagcct ctggattcac cttcagtaat catgctatgc actgggtccg ccaggctcca 180

ggcaaggggc tggagtgggt ggcagttata tcatatgatg gcgagaacac atattatgca 240

gactccattg agggccgatt caccatttcc agagacaatt tcaagaacac actctttcta 300

caaatgtaca gcctgacagc tgatgacacg gctatgtact tctgtgcgag agggggccgt 360

cgggggcact tcacctcata ctaccttgac tactggggcc agggaaccct ggtcaccgtc 420

tcctcggcta gtaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 480

tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga acccgtgacg 540

gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 600

tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 660

cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 720

gagcccaaat cttgtgacaa gactcacaca tgcccaccgt gcccagcacc tgaactcctg 780

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 960

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020

ggcaaggagt acaagtgcaa ggtttccaac aaagccctcc cagcccccat cgagaaaacc 1080

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1140

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1200

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260

cccgtgttgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1320

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1380

tacacgcaaa aaagcctctc cctgtctccc ggttga 1416

<210> 52

<211> 1359

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 52

caggtgcagc tggtggagtc tgggggaggc gtagtccagc ctgggaggtc cctgagactg 60

tcctgtgcag cctctggatt caccttcagt aatcatgcta tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggcgagaa cacatattat 180

gcagactcca ttgagggccg attcaccatt tccagagaca atttcaagaa cacactcttt 240

ctacaaatgt acagcctgac agctgatgac acggctatgt acttctgtgc gagagggggc 300

cgtcgggggc acttcacctc atactacctt gactactggg gccagggaac cctggtcacc 360

gtctcctcgg ctagtaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 420

acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaacccgtg 480

acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta 540

cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600

acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 660

gttgagccca aatcttgtga caagactcac acatgcccac cgtgcccagc acctgaactc 720

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960

aatggcaagg agtacaagtg caaggtttcc aacaaagccc tcccagcccc catcgagaaa 1020

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080

cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200

cctcccgtgt tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320

cactacacgc aaaaaagcct ctccctgtct cccggttga 1359

<210> 53

<211> 990

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 53

gctagtacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaacccgt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgtg acaagactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtttc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ttggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

caaaaaagcc tctccctgtc tcccggttga 990

<210> 54

<211> 369

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 54

caggtgcagc tggtggagtc tgggggaggc gtagtccagc ctgggaggtc cctgagactg 60

tcctgtgcag cctctggatt caccttcagt aatcatgcta tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggcgagaa cacatattat 180

gcagactcca ttgagggccg attcaccatt tccagagaca atttcaagaa cacactcttt 240

ctacaaatgt acagcctgac agctgatgac acggctatgt acttctgtgc gagagggggc 300

cgtcgggggc acttcacctc atactacctt gactactggg gccagggaac cctggtcacc 360

gtctcctcg 369

<210> 55

<211> 711

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 55

atggacatgc gggtgcccgc ccagctgctg ggcctgctgc tgctgtggtt ccccggctct 60

agatgcgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 120

gtcaccatca cttgccgggc aagccagaac atagacaagt acttaaattg gtatcagcag 180

ataccgggga aagcccctaa gctcctgatc tatgctgcat cgagtttgca cagtggggtc 240

ccatcaaggt tcagtggcag tggatctggg acagatttct ctctcaccat cagcagtctg 300

caacctgaag attttgcaat ttactactgt caacagagtt acagttcctt ccggacgttc 360

ggccaaggga ccaagctgga gatcaaacgt acggtggctg caccatctgt cttcatcttc 420

ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480

ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 540

tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600

ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660

cagggcctga gttcgcccgt cacaaagagc ttcaacaggg gagagtgttg a 711

<210> 56

<211> 645

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 56

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagcca gaacatagac aagtacttaa attggtatca gcagataccg 120

gggaaagccc ctaagctcct gatctatgct gcatcgagtt tgcacagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttctctctca ccatcagcag tctgcaacct 240

gaagattttg caatttacta ctgtcaacag agttacagtt ccttccggac gttcggccaa 300

gggaccaagc tggagatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagttcgc ccgtcacaaa gagcttcaac aggggagagt gttga 645

<210> 57

<211> 324

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 57

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagttcgcc cgtcacaaag 300

agcttcaaca ggggagagtg ttga 324

<210> 58

<211> 321

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polynucleotide "

<400> 58

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagcca gaacatagac aagtacttaa attggtatca gcagataccg 120

gggaaagccc ctaagctcct gatctatgct gcatcgagtt tgcacagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttctctctca ccatcagcag tctgcaacct 240

gaagattttg caatttacta ctgtcaacag agttacagtt ccttccggac gttcggccaa 300

gggaccaagc tggagatcaa a 321

<210> 59

<211> 19

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<400> 59

Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg

1 5 10 15

Val Leu Ser

<210> 60

<211> 22

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<400> 60

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys

<210> 61

<211> 37

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein (GA)15"

<400> 61

Cys His His His His His His Gly Ala Gly Ala Gly Ala Gly Ala Gly

1 5 10 15

Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly

20 25 30

Ala Gly Ala Gly Ala

<210> 62

<211> 31

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein (GP)15"

<400> 62

Cys Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly

1 5 10 15

Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro

20 25 30

<210> 63

<211> 31

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein (GR)15"

<400> 63

Cys Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly

1 5 10 15

Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg

20 25 30

<210> 64

<211> 31

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat Protein (PA)15"

<400> 64

Cys Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro

1 5 10 15

Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala

20 25 30

<210> 65

<211> 31

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat Protein (PR)15"

<400> 65

Cys Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg Pro

1 5 10 15

Arg Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg

20 25 30

<210> 66

<211> 30

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein peptide GA'

<400> 66

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

1 5 10 15

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

20 25 30

<210> 67

<211> 30

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein peptide GP "

<400> 67

Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro

1 5 10 15

Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro

20 25 30

<210> 68

<211> 30

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein peptide GR "

<400> 68

Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg

1 5 10 15

Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg

20 25 30

<210> 69

<211> 30

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein peptide PA'

<400> 69

Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala

1 5 10 15

Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala

20 25 30

<210> 70

<211> 30

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein peptide PR "

<400> 70

Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg

1 5 10 15

Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg Pro Arg

20 25 30

<210> 71

<211> 46

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein (GA)20"

<400> 71

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

1 5 10 15

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

20 25 30

Gly Ala Gly Ala Gly Ala Gly Ala His His His His His His

35 40 45

<210> 72

<211> 26

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein (GA)10"

<400> 72

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

1 5 10 15

Gly Ala Gly Ala His His His His His His

20 25

<210> 73

<211> 18

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein (GA)6"

<400> 73

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala His His His His

1 5 10 15

His His

<210> 74

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein (GA)5"

<400> 74

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala His His His His His His

1 5 10 15

<210> 75

<211> 14

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein (GA)4"

<400> 75

Gly Ala Gly Ala Gly Ala Gly Ala His His His His His His

1 5 10

<210> 76

<211> 12

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein (GA)3"

<400> 76

Gly Ala Gly Ala Gly Ala His His His His His His

1 5 10

<210> 77

<211> 10

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Dipeptide repeat protein (GA)2"

<400> 77

Gly Ala Gly Ala His His His His His His

1 5 10

<210> 78

<211> 10

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 78

Gly Gly Ser Val Ser Asp Tyr Tyr Trp Ser

1 5 10

<210> 79

<211> 20

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<400> 79

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

1 5 10 15

Gly Ala Gly Ala

<210> 80

<211> 12

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<400> 80

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

1 5 10

<210> 81

<211> 16

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Peptides "

<400> 81

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

1 5 10 15

<210> 82

<211> 40

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<400> 82

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

1 5 10 15

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

20 25 30

Gly Ala Gly Ala Gly Ala Gly Ala

35 40

<210> 83

<211> 30

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

Polypeptide "

<220>

<221> site

<222> (1)..(30)

<223 >/Note = "the sequence may include 6-15" Gly Ala "repeat units"

<400> 83

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

1 5 10 15

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

20 25 30

<210> 84

<211> 6

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<221> sources

<223 >/comment = "description of artificial sequence: synthesis of

6XHis tag "

<400> 84

His His His His His His

1 5

157页详细技术资料下载

上一篇：一种医用注射器针头装配设备

下一篇：SMO蛋白的特异性表位、识别该表位的抗体以及包含该抗体的组合物

Human anti- (poly-GA) dipeptide repeat (DPR) antibodies

相关技术

网友询问留言