RU2777844C1 - Antibody against beta-amyloid, antigen-binding fragment of said antibody, and application thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular, to an antibody against beta-amyloid (Abeta) or an antigen-binding fragment of said antibody, as well as to a composition containing it. Also disclosed are a nucleic acid molecule encoding the above antibody or a fragment thereof, as well as a cell and a vector containing it. Invention also relates to a method for producing an antibody against Abeta or an antigen-binding fragment thereof.

EFFECT: invention is effective for treating or preventing an Abeta-mediated disease or disorder.

19 cl, 15 dwg, 5 tbl, 4 ex

Description

Field of invention

The present disclosure belongs to the field of biotechnology. More specifically, the present disclosure relates to antibodies against β-amyloid and their uses.

Prior Art

The descriptions in this document are intended to provide background information on the present invention only and do not necessarily constitute prior art.

β-Amyloid (Amyloid-beta, beta-amyloid, Abeta, Aβ) is a polypeptide containing 39-43 amino acids formed from the amyloid precursor protein (APP - from the English amyloid precursor protein) as a result of proteolysis of β- and γ- secretases. It can be produced by a variety of cells and circulates in the blood, cerebrospinal fluid, and cerebral interstitial fluid. Most of the β-amyloid binds to chaperone molecules, and a small amount exists in the free state. Aβ is neurotoxic. It cannot be metabolized by cells when its content increases. On the contrary, it will accumulate in large quantities in cells with the formation of Aβ senile plaques and, as a result, stimulate damage and death of neurons. The most common Aβ subtypes in the human body are Aβ1-40 and Aβ1-42, of which Aβ1-42 is more toxic and more readily aggregates to form Aβ plaque cortex and causes neurotoxic effects (Medical Review, 2009, 15 (23): 3575 -3577).

Alzheimer's disease (AD - from the English. Alzheimer disease) was first discovered in 1906 by the German psychiatrist and neurologist, Alois Alzheimer, and was named after him. Also known as senile dementia, it is a chronic neurodegenerative disease. The main clinical manifestations of AD include gradual memory loss, cognitive impairment, abnormal behavior, and impaired social behavior. Studies have found that patients with AD mainly have the following pathological features: senile plaques formed as a result of aggregation of β-amyloid in the cerebral cortex and hippocampus, neurofibrillary tangles formed as a result of pathological aggregation of tau protein, and a reduced level of nerve cells in grief brain and hippocampus. Deposition of Aβ is not only associated with degenerative changes in neurons, but is also capable of activating a number of pathological events, including activation of astrocytes and microglial cells, destruction of the blood-brain barrier and changes in microcirculation, etc. This is the main cause of neuronal degeneration around senile plaques in the brain and death in AD patients (Oh, E.S., Troncoso, J.C. & Fangmark Tucker, S.M. Neuromol Med (2008) 10: 195).

Deposition of Aβ is found not only in senile plaques in Alzheimer's disease, but also found in large quantities in other cerebral amyloidosis, such as cerebral amyloid angiopathy (CAA - from the English cerebral amyloid angiopathy, also called congophilic amyloid angiopathy). The Aβ deposition found in CAA mainly consists of the shorter form of Aβ (Aβ1-40) and it also includes a small amount of Aβ1-42 (Wilcock et al., Journal of Neuroinflammation 1 (24):1-11 (2004) ).

Based on the important role of Aβ in the pathogenesis of AD and the fact that the toxicity of Aβ depends on its amount, degree of aggregation and clearance rate, researchers have long developed different ways to prevent diseases associated with Aβ, trying to delay or block the progression of AD by reducing the production of Aβ. , preventing the accumulation and deposition of Aβ and preventing the toxicity of Aβ. Numerous anti-Aβ antibodies have been published, including Aducanumab (Biogen), Bapineuzumab (Janssen, Elan, Pfizer), Solanezumab (Eli Lilly), Gantenerumab (Hoffman-LaRoche), and anti-Aβ antibodies disclosed in Patent Application No. WO 1994017197 A1, WO 1998044955 A1, WO 2003016466 A3, WO 2003070760 A3, WO 2004071408 A3, WO 2006081171 A1, WO 2007108756 A1, WO 2008011348 A3, WO 2008081008 A1, WO 20180514 etc. However, most antibodies have failed, including Bapineuzumab and Solanezumab, which showed great promise but failed in Phase III clinical trials. At present, only Aducanumab is still in a Phase III clinical trial and is believed to be the only new anti-Aβ antibody promising to be approved for the treatment of AD, however more results from clinical trials are still needed to confirm that whether it is suitable for sale.

Thus, there is still a strong need to develop novel structured anti-Abeta antibodies for the treatment of diseases or disorders caused by amyloid-beta (such as AD).

Brief summary of the invention

The inventors have developed anti-Abeta antibodies and their antigen-binding fragment with a completely new structure after extensive experimental studies. These anti-Abeta antibodies and their antigen-binding fragment are able to specifically bind to human Abeta with high affinity and exhibit excellent efficacy. They are expected to be useful in the treatment or prevention of diseases or disorders caused by Abeta (eg, Alzheimer's disease).

In some embodiments, the present disclosure provides an anti-Abeta antibody, or an antigen-binding fragment thereof, that specifically binds to human Abeta, said antibody comprising at least one CDR (complementary determining region) selected from the following amino acid sequences or having at least 85% sequence identity with them:

(1) the amino acid sequence of the heavy chain complementarity determining region (HCDR) of HCDR1 as shown in SEQ ID NO: 11,17 or 23;

(2) the amino acid sequence of the heavy chain HCDR2 region as shown in SEQ ID NO: 12, 18, 24, or 52;

(3) the amino acid sequence of the heavy chain HCDR3 region as shown in SEQ ID NO: 13, 19, 25, or 27;

(4) the amino acid sequence of the light chain complementarity determining region (LCDR) of LCDR1 as shown in SEQ ID NO: 14 or 20;

(5) the amino acid sequence of the light chain LCDR2 region as shown in SEQ ID NO: 15 or 21; and

(6) the amino acid sequence of the light chain LCDR3 region as shown in SEQ ID NO: 16, 22, 26, or 53.

In the case of an amino acid sequence having at least 85% sequence identity as above, it preferably has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; more preferably it has greater than 90%, greater than 95% or greater than 99% sequence identity, most preferably it has at least 95% sequence identity. The above amino acid sequence having at least 85% sequence identity can be obtained by deletion, insertion or substitution of one or more amino acids.

In some embodiments, said anti-Abeta antibody binds to human Aβ1-42 fibrils and/or human Aβ1-42 monomer with a dissociation equilibrium constant (KD) of about 10 ^-7 M or even less; preferably binds to human Abeta with a dissociation equilibrium constant (KD) of less than about 10 ^-8 M, 10 ^-9 M or 10 ^-10 M or even less. The KD value can be determined using surface plasmon resonance (SPR) technology on a Biacore T200 instrument. Preferably, said human Abeta is Aβ1-42 fibrils and Aβ1-42 monomer.

In some preferred embodiments, said anti-Abeta antibody, or antigen-binding fragment thereof, comprises HCDR1-3 regions or LCDR1-3 regions as specified in any of the following:

(i) the HCDR1, HCDR2 and HCDR3 regions of the antibody heavy chain as shown in the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively;

(ii) the HCDR1, HCDR2 and HCDR3 regions of the antibody heavy chain as shown in the amino acid sequences of SEQ ID NOs: 17, 18 and 19, respectively;

(iii) the HCDR1, HCDR2 and HCDR3 regions of the antibody heavy chain as shown in the amino acid sequences of SEQ ID NOs: 23, 24 and 25, respectively;

(iv) the HCDR1, HCDR2 and HCDR3 regions of the antibody heavy chain as shown in the amino acid sequences of SEQ ID NOs: 17, 18 and 27, respectively;

(v) the HCDR1, HCDR2 and HCDR3 regions of the antibody heavy chain as shown in the amino acid sequences of SEQ ID NOs: 17, 52 and 19, respectively;

(vi) the HCDR1, HCDR2 and HCDR3 regions of the antibody heavy chain as shown in the amino acid sequences of SEQ ID NOs: 17, 52 and 27, respectively;

and/or

(vii) the LCDR1, LCDR2 and LCDR3 regions of the antibody light chain as shown in the amino acid sequences of SEQ ID NOs: 14, 15 and 16, respectively;

(viii) the LCDR1, LCDR2 and LCDR3 regions of the antibody light chain as shown in the amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively;

(ix) the LCDR1, LCDR2 and LCDR3 regions of the antibody light chain as shown in the amino acid sequences of SEQ ID NOs: 20, 21 and 26, respectively; or

(x) the LCDR1, LCDR2 and LCDR3 regions of the antibody light chain as shown in the amino acid sequences of SEQ ID NOS: 20, 21 and 53, respectively.

In some embodiments, an anti-Abeta antibody, or antigen-binding fragment thereof, of the present disclosure is any anti-Abeta antibody, or antigen-binding fragment thereof, selected from the following A through D:

A) an anti-Abeta antibody or antigen-binding fragment thereof, having a heavy chain HCDR1 region as shown in the amino acid sequence of SEQ ID NO 11, a heavy chain HCDR2 region as shown in the amino acid sequence of SEQ ID NO 12, and a heavy chain HCDR3 region as shown in the amino acid sequence of SEQ ID NO 13, and the light chain LCDR1 region as shown in the amino acid sequence of SEQ ID NO 14, the light chain LCDR2 region as shown in the amino acid sequence of SEQ ID NO 15, and the light chain LCDR3 region as shown in the amino acid sequence of SEQ ID NO 16;

B) an anti-Abeta antibody or antigen-binding fragment thereof, having a heavy chain HCDR1 region as shown in the amino acid sequence of SEQ ID NO 17, a heavy chain HCDR2 region as shown in the amino acid sequence of SEQ ID NO: 18 or 52, and a heavy chain HCDR3 region, as shown in the amino acid sequence of SEQ ID NO 19, and the light chain LCDR1 region as shown in the amino acid sequence of SEQ ID NO 20, the light chain LCDR2 region as shown in the amino acid sequence of SEQ ID NO 21, and the light chain LCDR3 region as shown in the amino acid sequence of SEQ ID NO 22;

C) an anti-Abeta antibody, or an antigen-binding fragment thereof, having a heavy chain HCDR1 region as shown in the amino acid sequence of SEQ ID NO 23, a heavy chain HCDR2 region as shown in the amino acid sequence of SEQ ID NO 24, and a heavy chain HCDR3 region as shown in the amino acid sequence of SEQ ID NO 25, and the light chain LCDR1 region as shown in the amino acid sequence of SEQ ID NO 20, the light chain LCDR2 region as shown in the amino acid sequence of SEQ ID NO 21, and the light chain LCDR3 region as shown in the amino acid sequence of SEQ ID NO: 26 or 53; and

D) an anti-Abeta antibody, or an antigen-binding fragment thereof, having a heavy chain HCDR1 region as shown in the amino acid sequence of SEQ ID NO 17, a heavy chain HCDR2 region as shown in the amino acid sequence of SEQ ID NO: 18 or 52, and a heavy chain HCDR3 region, as shown in the amino acid sequence of SEQ ID NO 27, and the light chain LCDR1 region as shown in the amino acid sequence of SEQ ID NO 20, the light chain LCDR2 region as shown in the amino acid sequence of SEQ ID NO 21, and the light chain LCDR3 region as shown in amino acid sequence of SEQ ID NO 22.

In some embodiments, said anti-Abeta antibody, or antigen-binding fragment thereof, is a murine, chimeric, humanized, or fully human antibody.

In some embodiments, said anti-Abeta antibody or antigen-binding fragment thereof is a murine antibody and the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 3, 5, 7, or 9 or has at least 85% sequence identity therewith. ; and/or

the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 4, 6, 8 or 10, or having at least 85% sequence identity with them.

In the case of an amino acid sequence having at least 85% sequence identity as above, it preferably has at least 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; more preferably it has greater than 90%, greater than 95% or greater than 99% sequence identity, most preferably it has at least 95% sequence identity. The above amino acid sequence having at least 85% sequence identity can be obtained by deletion, insertion or substitution of one or more amino acids. Preferably, said antibody binds to human Abeta with a dissociation equilibrium constant (KD) of about 10 ^-7 M or even less. In some embodiments, said antibody binds to human Abeta with a dissociation equilibrium constant (KD) of less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or even less, and the KD value can be determined using Surface Plasmon Resonance (SPR) technology. , from the English Surface Plasma Resonance) on the Biacore T200 device. Preferably, said human Abeta is Aβ1-42 fibrils and Aβ1-42 monomer.

In some embodiments, said anti-Abeta antibody, or antigen-binding fragment thereof, is any anti-Abeta antibody, or antigen-binding fragment thereof, selected from the following E to H:

E) an anti-Abeta antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO 3 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO 4;

F) an anti-Abeta antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO 5 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO 6;

G) an anti-Abeta antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO 7 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO 8; and

H) an anti-Abeta antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO 9 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO 10.

In some embodiments of the aforementioned anti-Abeta antibody or antigen-binding fragment thereof of the present disclosure, said antibody is a humanized antibody. Preferably, the FR sequence (from the English framework region - frame region) of the antibody region comes from the sequence of the FR region of the human germline antibody or its variant. Preferably, if necessary, this option includes one or more deletions, insertions or substitutions of amino acids; More preferably, the FR region variant has up to 10 amino acid backmutations in the human antibody light chain framework and/or heavy chain framework, respectively.

In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-Abeta antibody, or antigen-binding fragment thereof, is shown in SEQ ID NOs: 44, 46, 48, or 50, or having at least 85% sequence identity therewith; and/or

the amino acid sequence of the light chain variable region of the anti-Abeta antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 45, 47, 49 or 51, or having at least 85% sequence identity with them.

In the case of an amino acid sequence having at least 85% sequence identity as above, it preferably has at least 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; more preferably it has greater than 90%, greater than 95% or greater than 99% sequence identity, most preferably it has at least 95% sequence identity. The above amino acid sequence having at least 85% sequence identity can be obtained by deletion, insertion or substitution of one or more amino acids. Preferably, said antibody binds to human Abeta with a dissociation equilibrium constant (KD) of about 10 ^-7 M or even less. In some embodiments, said antibody binds to human Abeta with a dissociation equilibrium constant (KD) of less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or even less, and the KD value can be determined using Surface Plasmon Resonance (SPR) technology. ) on the Biacore T200 instrument. Preferably, said human Abeta is Aβ1-42 fibrils and Aβ1-42 monomer.

In some embodiments, said antibody is a humanized antibody selected from any of the anti-Abeta antibodies or antigen-binding fragment thereof of the following a) to d):

a) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody has at least 90% sequence identity with the sequence as shown in SEQ ID NO 44, and the light chain variable region of the antibody has at least 90% multiple sequence identity with the sequence as shown in SEQ ID NO 45;

b) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody has at least 90% sequence identity with the sequence as shown in SEQ ID NO 46, and the light chain variable region of the antibody has at least 90% multiple sequence identity with the sequence as shown in SEQ ID NO 47;

c) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody has at least 90% sequence identity with the sequence as shown in SEQ ID NO 48, and the light chain variable region of the antibody has at least 90% multiple sequence identity with the sequence as shown in SEQ ID NO 49; and

d) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody has at least 90% sequence identity with the sequence as shown in SEQ ID NO 50, and the light chain variable region of the antibody has at least 90% sequence identity with a sequence as shown in SEQ ID NO 51, where the above at least 90% sequence identity includes at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, said antibody is a humanized antibody containing variable regions as shown in any of the following:

a) an antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO 11, SEQ ID NO 12 and SEQ ID NO 13, respectively, and FR region(s) containing(s) one or more amino acids back mutations selected from the group consisting of 1E, 71A and 94R; and an antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, respectively;

b) an antibody heavy chain variable region containing HCDR1 and HCDR3, as shown in SEQ ID NO 17 and SEQ ID NO 19, respectively, and HCDR2, as shown in SEQ ID NO: 18 or 52, and the FR region(s) containing (i) one or more amino acid backmutations selected from the group consisting of 28A and 82B T; an antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, respectively;

c) an antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO 23, SEQ ID NO 24 and SEQ ID NO 25, respectively; and an antibody light chain variable region comprising LCDR1 and LCDR2 as shown in SEQ ID NO 20 and SEQ ID NO 21, respectively, and LCDR3 as shown in SEQ ID NO: 26 or 53, and FR region(s) containing ( f) one or more amino acid backmutations selected from the group consisting of 2V and 45K; and

d) an antibody heavy chain variable region comprising HCDR1 and HCDR3 as shown in SEQ ID NO 17 and SEQ ID NO 27, respectively, and HCDR2 as shown in SEQ ID NO: 18 or 52; and an antibody light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO 20, SEQ ID NO 21, and SEQ ID NO 22, respectively, and FR region(s) containing(s) amino acid backmutation 2V.

In some embodiments, said antibody is a humanized antibody containing variable regions as shown in any of the following:

a) an antibody heavy chain variable region as shown in the amino acid sequence of SEQ ID NO 66, or a FR region variant thereof; and an antibody light chain variable region as shown in the amino acid sequence of SEQ ID NO 67, or a FR region variant thereof;

b) an antibody heavy chain variable region as shown in the amino acid sequence of SEQ ID NO 68, or a FR region variant thereof; and an antibody light chain variable region as shown in the amino acid sequence of SEQ ID NO 69, or a FR region variant thereof;

c) an antibody heavy chain variable region as shown in the amino acid sequence of SEQ ID NO 70, or a FR region variant thereof; and an antibody light chain variable region as shown in the amino acid sequence of SEQ ID NO 71, or a FR region variant thereof; and

d) an antibody heavy chain variable region as shown in the amino acid sequence of SEQ ID NO 72, or a FR region variant thereof; and an antibody light chain variable region as shown in the amino acid sequence of SEQ ID NO 73, or a FR region variant thereof; wherein the FR region variant has up to 10 amino acid backmutations separately in the light chain framework region and/or the heavy chain framework region.

In some embodiments, the above-mentioned anti-Abeta antibody or antigen-binding fragment thereof according to the present disclosure is any anti-Abeta antibody or antigen-binding fragment selected from the following M - P:

M) an anti-Abeta antibody or antigen-binding fragment thereof, comprising a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO 44 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO 45;

N) an anti-Abeta antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO 46 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO 47;

O) an anti-Abeta antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO 48 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO 49;

P) an anti-Abeta antibody or antigen-binding fragment thereof, comprising a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO 50 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO 51.

In some embodiments, said anti-Abeta antibody, or antigen-binding fragment thereof, further comprises a constant region.

In some embodiments, the antibody heavy chain constant region is a human IgG1 constant region, preferably, the amino acid sequence of the antibody heavy chain constant region is as shown in SEQ ID NO 42 or has at least 85% sequence identity with it; and the antibody light chain constant region is selected from a human kappa constant region, and most preferably the amino acid sequence of the antibody light chain constant region is as defined in SEQ ID NO 43 or has at least 85% sequence identity with it.

In the case of an amino acid sequence having at least 85% sequence identity as above, it preferably has at least 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; more preferably it has greater than 90%, greater than 95% or greater than 99% sequence identity, most preferably it has at least 95% sequence identity. The above amino acid sequence having at least 85% sequence identity can be obtained by deletion, insertion or substitution of one or more amino acids.

In some embodiments, the amino acid sequence of the heavy chain of an anti-Abeta antibody is as shown in SEQ ID NO: 30, 34, 38, or 40, or has at least 85% sequence identity therewith, and/or

the amino acid sequence of the light chain of the anti-Abeta antibody is shown in SEQ ID NO: 31, 35, 39 or 41, or having at least 85% sequence identity with them.

In the case of an amino acid sequence having at least 85% sequence identity as above, it preferably has at least 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; more preferably it has greater than 90%, greater than 95% or greater than 99% sequence identity, most preferably it has at least 95% sequence identity. The above amino acid sequence having at least 85% sequence identity can be obtained by deletion, insertion or substitution of one or more amino acids. Preferably, said antibody binds to human Abeta with a dissociation equilibrium constant (KD) of about 10 ^-7 M or even less. In some embodiments, said antibody binds to human Abeta with a dissociation equilibrium constant (KD) of less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or even less, and the KD value can be determined using Surface Plasmon Resonance (SPR) technology. ) on the Biacore T200 instrument. Preferably, said human Abeta is Aβ1-42 fibrils and Aβ1-42 monomer.

In some embodiments, said anti-Abeta antibody, or antigen-binding fragment thereof, is any anti-Abeta antibody, or antigen-binding fragment thereof, selected from the following Q-T:

Q) an anti-Abeta antibody, or antigen-binding fragment thereof, comprising a heavy chain as shown in the amino acid sequence of SEQ ID NO 30 and a light chain as shown in the amino acid sequence of SEQ ID NO 31;

R) an anti-Abeta antibody or antigen-binding fragment thereof, comprising a heavy chain as shown in the amino acid sequence of SEQ ID NO 34 and a light chain as shown in the amino acid sequence of SEQ ID NO 35;

S) an anti-Abeta antibody, or antigen-binding fragment thereof, comprising a heavy chain as shown in the amino acid sequence of SEQ ID NO 38 and a light chain as shown in the amino acid sequence of SEQ ID NO 39; and

T) an anti-Abeta antibody or antigen-binding fragment thereof, comprising a heavy chain as shown in the amino acid sequence of SEQ ID NO 40 and a light chain as shown in the amino acid sequence of SEQ ID NO 41.

In some embodiments, the antigen-binding fragment of the present disclosure is selected from the group consisting of Fab (from the English antigen-binding fragment - antigen-binding fragment), Fab', F(ab') ₂ , scFv (from the English single-chain variable fragment - single-chain variable fragment), diabody, dsFv, and an antigen-binding fragment of a peptide containing a CDR.

In some embodiments, an anti-Abeta antibody is provided that competes with the aforementioned antibody or antigen-binding fragment thereof for binding to human Abeta, or competes with the aforementioned antibody or antigen-binding fragment for binding to the same Abeta antigen epitope.

In some embodiments, the present disclosure also provides a nucleic acid molecule encoding any anti-Abeta antibody, or antigen-binding fragment thereof, as described above.

In some embodiments, the nucleic acid molecule is any nucleic acid molecule selected from the following U - Z:

U) a nucleic acid molecule that contains a nucleotide sequence as shown in SEQ ID NO 52, or a nucleotide sequence having at least 85% sequence identity with it, and/or contains a nucleotide sequence as shown in SEQ ID NO 53 , or a nucleotide sequence having at least 85% sequence identity with it;

V) a nucleic acid molecule that contains a nucleotide sequence as shown in SEQ ID NO 54, or a nucleotide sequence having at least 85% sequence identity with it, and/or contains a nucleotide sequence as shown in SEQ ID NO 55 , or a nucleotide sequence having at least 85% sequence identity with it;

W) a nucleic acid molecule that contains a nucleotide sequence as shown in SEQ ID NO 56, or a nucleotide sequence having at least 85% sequence identity with it, and/or contains a nucleotide sequence as shown in SEQ ID NO 57 , or a nucleotide sequence having at least 85% sequence identity with it; and

Z) a nucleic acid molecule that contains a nucleotide sequence as shown in SEQ ID NO 58, or a nucleotide sequence having at least 85% sequence identity with it, and/or contains a nucleotide sequence as shown in SEQ ID NO 59 , or a nucleotide sequence having at least 85% sequence identity with it.

In one aspect, the present disclosure also provides a recombinant vector that contains the nucleic acid molecule mentioned above.

In one aspect, the present disclosure also provides a host cell obtained by transformation with the recombinant vector mentioned above; the host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells, including CHO (Chinese Hamster ovary cell line) and NS0 cells.

In yet another aspect, the present disclosure provides a method for producing the aforementioned anti-Abeta antibody or antigen-binding fragment thereof, including the steps of culturing the aforementioned host cell in a culture medium and then purifying and isolating the antibody.

In yet another aspect, the present disclosure also provides a pharmaceutical composition comprising the above-mentioned anti-Abeta antibody or antigen-binding fragment thereof and one or more pharmaceutically acceptable carriers, excipients or diluents.

In yet another aspect, the present disclosure also provides the use of the aforementioned anti-Abeta antibody or antigen-binding fragment thereof or the aforementioned pharmaceutical composition in the preparation of a medicament for the treatment or prevention of a disease or disorder caused by Abeta.

In yet another aspect, the present disclosure also provides a method of treating or preventing a disease or disorder caused by Abeta, comprising administering a therapeutically effective amount of the aforementioned anti-Abeta antibody or antigen-binding fragment thereof or the aforementioned pharmaceutical composition to a patient having the disease or disorder.

In yet another aspect, the present disclosure also provides the aforementioned anti-Abeta antibody or antigen-binding fragment thereof, or the aforementioned pharmaceutical composition for use as a medicament for the treatment of Abeta-mediated diseases or disorders.

In some embodiments, the disease or disorder mentioned above is a neurodegenerative disease selected from the group consisting of Alzheimer's disease, mild cognitive impairment, frontotemporal dementia, Lewy body dementia, Parkinson's disease, Pick's disease, Bewak's disease, congophilic amyloid angiopathy, cerebral amyloid angiopathy , Down syndrome, multi-infarct dementia, Huntington's disease, Creutzfeldt-Jakob disease, AIDS dementia syndrome, depression, anxiety neurosis, phobia, Bell's palsy, epilepsy, encephalitis, multiple sclerosis, neuromuscular disorder, neuro-oncological disease, brain tumor, neurovascular disorders due to stroke, neuroimmunological disease, neuropathic otological disease, traumatic injury to the nervous system due to spinal cord injury, pain due to neuropathic pain, childhood neuro- and neuropsychiatric disorder disorders, sleep disorders, Tourette syndrome, mild cognitive impairment, vascular dementia, multi-infarct dementia, cystic fibrosis, Gaucher disease and other dyskinesias, and diseases of the central nervous system. In some embodiments, the disease is Alzheimer's disease.

In some embodiments, the present disclosure also provides a method of treating or preventing Alzheimer's disease, comprising administering to a patient a therapeutically effective amount of the aforementioned anti-Abeta antibody or antigen-binding fragment thereof, or the aforementioned pharmaceutical composition.

The anti-Abeta antibodies or antigen-binding fragments thereof of the present disclosure demonstrate good performance in both biochemical assays and in vivo pharmacodynamic assays. For example, in an affinity detection assay, the anti-Abeta antibodies of the present disclosure (HAB-2401, HAB-5101, HAB-3601, and HAB-9001) show an affinity for Aβ1-42 fibrils of less than 10 ^-8 M, even less than 10 ^-10 M, and the affinity for the monomer Aβ1-42 is less than 10 ^-7 M - 10 ^-8 M. In particular, the HAB-9001 antibody shows an affinity almost 100 times stronger than Aducanumab, currently known as the most excellent anti-Abeta antibody. .

In addition, in the analysis of the effect of antibodies on blocking the aggregation of Aβ1-42 monomers into Aβ1-42 fibrils, the results show that the antibodies HAB-2401, HAB-5101 and HAB-9001 of the present disclosure can effectively inhibit the aggregation of Aβ1-42 monomer into Aβ1 fibrils. -42 whereas Aducanumab cannot.

In biochemical analysis, the results show that the anti-Abeta antibodies HAB-2401 and HAB-3601 of the present disclosure have an impact on the protection of primary rat neurons and can stimulate phagocytosis of Aβ fibrils by primary microglial cells.

In addition, the results of pharmacodynamic analysis in animals show that the antibodies HAB-2401, HAB-5101 and HAB-9001 of the present disclosure can significantly improve the spatial memory and cognitive ability of 5*FAD mice with Alzheimer's disease. In addition, the antibodies of the present disclosure have a weaker effect on the release of cytokines (such as TNFα (tumor necrosis factor alpha), IFN-γ (from the English interferon gamma - interferon gamma), IL1p (from the English. Interleukin - interleukin), IL2, IL6, IL12p70, IL4, IL10, MCP-1 (from the English Monocyte Chemotactic Protein 1 - Monocytic chemotactic protein 1) and KS) in brain tissue, compared with a positive control - the Adukanumab antibody, which indicates that the antibodies of the present disclosure are controlled in terms of safety hazards. Anti-Abeta antibodies of the present disclosure are expected to be useful in the future for the treatment or prevention of diseases or disorders caused by Abeta (such as Alzheimer's disease).

Description of graphic materials

On FIG. 1 shows a fluorescence analysis of thioflavin (ThT) showing the results of experiments on the blocking effect of anti-Abeta antibodies on the aggregation of Aβ1-42 monomers into Aβ1-42 fibrils;

On FIG. 2 depicts a protection assay on primary cortical neurons showing the results of experiments on the protective effect of anti-Abeta antibodies on rat primary neurons;

On FIG. 3 is a BV2 phagocytosis assay showing the results of experiments on anti-Abeta antibodies that stimulate phagocytosis of Aβ fibrils by BV2 cells;

On FIG. 4 shows the results of experiments on the effects of anti-Abeta antibodies on phagocytosis of Aβ fibrils by primary rat microglial cells;

Fig. 5 is a representative scoring picture of the ability of mice to build a nest;

On FIG. 6 shows a nesting experiment with a bar graph showing the effect of anti-Abeta antibodies on improving the social behavior of 5xFAD transgenic mice;

On FIG. 7 shows a nesting experiment, with a scatterplot showing the effect of anti-Abeta antibodies on improving the social behavior of 5xFAD transgenic mice;

On FIG. 8 shows a schematic representation of a water maze. The solid circle and dashed circle represent the location of the platform and platform area, respectively.

On FIG. 9 shows the learning curves for the water maze experiment; shows the results of training and learning curves with a hidden platform. Two-way analysis of variance refers to two-way analysis of variance; The analysis results look like this:

Two-way analysis of variance: WT (from the English. wild type - wild type) in comparison with the Carrier: # p less than 0.05; ###: p less than 0.001;

меньше 0,05;

р меньше 0,01;

р меньше 0,001;Amab vs Carrier:

less than 0.05;

p is less than 0.01;

p is less than 0.001;

HAB-5101-Ch vs Vehicle: ^: p less than 0.05;

р меньше 0,05;

р меньше 0,01;NAV-2401-Ch in comparison with the Carrier:

p less than 0.05;

p is less than 0.01;

HAB-9001-Ch compared to Vehicle: *: p less than 0.05; **: p less than 0.01; ***: P less than 0.001.

On FIG. 10A-10G depict the results of the water maze experiment: the horizontal axis shows drug administration groups; On FIG. 10A shows the results for the delay time in reaching the platform, FIG. 10B shows results for delay time in reaching the platform area, FIG. 10C shows the platform crossing count results, FIG. 10D shows the results of the platform area crossing count, FIG. 10E shows the results for platform dwell time, FIG. 10F shows the results for the length of stay in the area of the platform, and FIG. 10G shows the results of the platform intersection index (the intersection index is the number of intersections of the target area - (the sum of the number of intersections of the corresponding areas in the other three sectors)/3), the legends of FIG. 10B-10G are the same as the legends of FIG. 10A (see legend in FIG. 10A for details). One-way analysis of variance refers to one-way analysis of variance (same in the case of Figs. 12 and 13), in comparison with the vehicle, *p less than 0.05, **p less than 0.01, ***p less than 0.001; no significant differences between groups were found;

On FIG. 11A-11D depict the results of an enzyme-linked immunosorbent assay (ELISA) of Aβ deposits. On FIG. 11A shows the content of insoluble Aβ1-40 present in the hippocampus, FIG. 11B shows the content of insoluble Aβ1-42 present in the hippocampus (the legend of FIG. 11B is the same as the legend of FIG. 11A, see FIG. 11A for more details), FIG. 11C shows the content of insoluble Aβ1-40 found in the cerebral cortex, and FIG. 11D shows the content of insoluble Aβ1-42 found in the cerebral cortex (the legend of Fig. 11D is the same as the legend of Fig. 11C, see Fig. 11C for more details);

Fig. 12 is a graph showing Aβ in the hippocampus showing the results of immunohistochemistry (IHC) - detection of Aβ deposition (cerebral cortex). One-way analysis of variance, compared to vehicle ***p less than 0.001;

On FIG. 13 shows Aβ in the cerebral cortex showing the results of IHC detection of Aβ deposition (hippocampus);

On FIG. 14A shows the effect of anti-Abeta antibodies on the release of a cytokine, TNFα, in brain tissue;

On FIG. 14B shows the effect of anti-Abeta antibodies on the release of the cytokine IFN-γ in brain tissue;

On FIG. 14C shows the effect of anti-Abeta antibodies on MCP-1 release in brain tissue;

On FIG. 14D shows the effect of anti-Abeta antibodies on CS release in brain tissue;

On FIG. 15A shows the effect of anti-Abeta antibodies on IL1γ cytokine release in brain tissue;

On FIG. 15B shows the effect of anti-Abeta antibodies on IL2 cytokine release in brain tissue;

On FIG. 15C shows the effect of anti-Abeta antibodies on IL6 cytokine release in brain tissue;

On FIG. 15D shows the effect of anti-Abeta antibodies on the release of the cytokine IL12p70 in brain tissue;

On FIG. 15E shows the effect of anti-Abeta antibodies on IL4 cytokine release in brain tissue;

On FIG. 15F shows the effect of anti-Abeta antibodies on IL10 cytokine release in brain tissue.

Terminology

For an easier understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used in this document have the meanings commonly implied by those skilled in the art to which this disclosure belongs.

Three-letter codes and one-letter codes for amino acids used in the present disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

In this disclosure, the term "XXX" refers to one or more substances; for example, the term "anti-Abeta antibody" should be understood as one or more antibodies that specifically bind to Abeta. Thus, the terms "one", "one or more", and "at least one" may be used interchangeably throughout this document.

The terms "amyloid β", "amyloid β", "Ap" and "Abeta" may be used interchangeably herein and refer to a segment formed by cleavage of the amyloid precursor protein (APP) using p-secretase 1 (BACE1), or any modification thereof or any functional equivalent, including but not limited to Aβ1-40 and Aβ1-42. It is known that Aβ is in the form of monomers, which are combined to form oligomers and a protofibrillar structure. The structure and sequences of such Aβ peptides are well known to those skilled in the art, and methods for preparing these peptides or isolating these peptides from the brain and other tissues are also well known (e.g., Glenner and Wong, Biochem Biophys Res. Comm. 129:885-890 ( 1984)). In addition, Aβ peptides are also commercially available. As examples of the amino acid sequence of human Aβ, SEQ ID NO 1 represents the amino acid sequence of Aβ1-40 and SEQ ID NO 2 represents the amino acid sequence of Aβ1-42.

As used herein, the term "antibody" refers to an immunoglobulin, a structure of four peptide chains linked together via a disulfide bond between two identical heavy chains and two identical light chains. Different immunoglobulin heavy chain constant regions exhibit different amino acid compositions and locations, hence representing different antigenicity. Accordingly, immunoglobulins can be classified into five types or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, according to the heavy chain μ, δ, γ, α and ε, respectively. According to its amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds, the same lg type can be further subdivided into different subtypes, for example, IgG can be subdivided into IgG1, IgG2, IgG3 and IgG4. The light chain can be subdivided into a κ or λ chain considering different constant region(s). Each of the five lg types can have a chain κ or λ.

In the present disclosure, an antibody light chain may further comprise a light chain constant region that contains a human or mouse κ, λ chain, or a variant thereof; the heavy chain of the antibody may further comprise a heavy chain constant region that contains human or mouse IgG1, IgG2, IgG3, IgG4, or a variant thereof.

Sequences of about 110 amino acids adjacent to the N-terminus of the heavy and light chains of an antibody are highly variable, known as the variable region (Fv region), including the light chain variable region (VL) and the heavy chain variable region (VH); the remaining amino acid sequences near the C-terminus are relatively stable, known as the constant region. The variable region includes three hypervariable regions (HVR - from the English hypervariable region) and four relatively conservative framework regions (FR). These three hypervariable regions, which determine the specificity of an antibody, are also known as complementarity determining regions (CDRs). Each light chain variable region (VL) and each heavy chain variable region (VH) consists of three CDR regions and four FR regions, with the sequential order from N-terminus to C-terminus being: FR1, CDR1, FR2, CDR2, FR3 , CDR3 and FR4. The three light chain CDR regions are LCDR1, LCDR2 and LCDR3; and the three heavy chain CDR regions are HCDR1, HCDR2 and HCDR3. The number and position of the amino acid residues of the CDRs of an antibody or antigen-binding fragment described herein conform to known Kabat numbering criteria and/or Chothia numbering criteria ((1983) U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest.").

Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.

The term "mouse antibody" described in this disclosure refers to a monoclonal antibody that binds human Abeta, obtained in accordance with knowledge and skill in this field. During preparation, the subject may be injected with the Abeta antigen and then isolated from a hybridoma expressing an antibody that has the desired sequence or functionality. In one embodiment of the present disclosure, the mouse anti-Abeta antibody, or antigen-binding fragment thereof, further comprises a mouse κ, λ light chain constant region, or a variant thereof, and/or further comprises a mouse IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a variant thereof.

The term "chimeric antibody", as described herein, is an antibody that is formed by fusing the variable region of a mouse antibody together with the constant region of a human antibody, and this chimeric antibody can facilitate an immune response elicited by the mouse antibody. To construct a chimeric antibody, a mouse-specific monoclonal antibody-secreting hybridoma is first created, and then the variable region gene is cloned from the mouse hybridoma. Then, the constant region gene is cloned from the human antibody as needed. A mouse variable region gene is fused to a human constant region gene to form a chimeric gene that can subsequently be inserted into an expression vector. Finally, the chimeric antibody molecule will be expressed in a eukaryotic or prokaryotic system. In one embodiment of the present disclosure, the light chain of the chimeric antibody further comprises a light chain constant region derived from the k, R, human chain, or a variant thereof. The heavy chain of the chimeric antibody further comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3, IgG4, or a variant thereof, preferably contains a heavy chain constant region derived from human IgG1, IgG2, or IgG4, or an IgG1, IgG2, or IgG4 variant with amino acid(s) mutation(s), such(s) as mutation(s) YTE or reverse(s) mutation(s).

"Humanized antibody", as used herein, also known as a CDR-grafted antibody, refers to an antibody generated by grafting murine CDR sequences into the variable region framework regions of a human antibody, namely an antibody derived from different human germline antibody framework sequence types. A humanized antibody can overcome the heterologous responses elicited by a chimeric antibody that carries a large amount of murine protein components. Such framework sequences can be obtained from a publicly available DNA database covering germline antibody gene sequences, or from published reference sequences. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the VBase human germline sequence database (www.mrccpe.com.ac.uk/vbase) and also in Kabat, EA, et al, 1991 Sequences of Proteins of Immunological Interest, 5th Ed. In order to avoid reduced activity caused by reduced immunogenicity, framework sequences in the variable region of a human antibody may undergo minimal backmutations or backmutation(s) to maintain activity. The humanized antibody of the present disclosure also includes a humanized antibody on which CDR affinity maturation is performed by phage display. In one embodiment of the present disclosure, the CDR sequence of a humanized anti-Abeta antibody is selected from SEQ ID NOs: 11-27. In the case of a human antibody variable region framework, after construction and selection, the FR sequence of the antibody heavy chain region is selected from the FR1, FR2, FR3 and JH6 human germline regions IGHV1-24*01 and hjh6.3, IGHV3-30*01 and hjh6 .3 or IGHV2-70D*04 and hjh6.1, or their mutant sequence; light chain FR region sequence selected from FR1, FR2, FR3 and JK4, JK2 human germline regions IGKV1-27*01 and hjk4.1, IGKV1-39*01 and hjk4.1, IGKV2-40*01 and hjk4.1, IGKV2-40*01 and hjk2.1 or their mutant sequence.

Grafting of the CDR may result in a decrease in the affinity of the resulting anti-Abeta antibody or antigen-binding fragment thereof for the antigen due to framework residues in contact with the antigen. Such interactions may be the result of highly somatic mutations. Thus, it may still be necessary to graft donor amino acid framework regions onto humanized antibody framework regions. Antigen-binding amino acid residues derived from a non-human anti-Abeta antibody or antigen-binding fragment thereof can be identified by checking the sequence and structure of the variable region of a mouse monoclonal antibody. Amino acid residues that differ between donor CDR and germline framework regions can be considered related. If it is not possible to determine the most closely related germline, the sequence can be compared to a common sequence shared by subtypes, or a mouse sequence with a high percentage of similarity. The rare framework residues are believed to be the result of a high level of mutation in somatic cells and play an important role in binding.

As used herein, the term "antigen-binding fragment" or "functional fragment" refers to one or more antibody fragments that retain the ability to bind to an antigen. It has been shown that fragments of a full-length antibody can be used to achieve the function of binding to a specific antigen. Examples of binding fragments encompassed by the term "antigen binding fragment" include: (i) Fab fragment, a monovalent fragment consisting of a VL domain, VH, CL and CH1; (ii) P(ab') ₂ fragment, divalent fragment containing two Fab-fragment connected by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VH and VL domains of one arm; (v) one dAb domain or fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain; and (vi) a single complementarity determining region (CDR), or (vii) a combination of two or more separate CDRs, optionally linked by a synthetic linker. In addition, the VL domain and the VH domain of the Fv fragment are encoded by two separate genes, however, they can be linked by a synthetic linker using genetic engineering methods to form a single protein chain, where a monovalent molecule is formed by pairing the VL and VH domains (referred to as single-stranded Fv (scFv) see for example Bird et al (1988): 423-426 Science 242 and Huston et al (1988) Proc Natl Acad Sci USA 85: 5879-5883). Such single chain antibodies are also intended to be included in the term "antigen binding fragment". Such antibodies are prepared using conventional techniques known in the art and screened for functional fragments using the same method as for an intact antibody. Antigen-binding portions can be obtained by genetic engineering or by enzymatic or chemical damage to intact immunoglobulin. Antibodies can be in the form of different isotypes, for example, IgG antibodies (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM.

As used herein, a Fab is an antibody fragment obtained by treating an IgG antibody molecule with papain (which cleaves off the amino acid residue at position 224 of the H chain). The Fab fragment has a molecular weight of about 50,000 and has antigen-binding activity where about half of the N-terminal side of the H chain and the entire L chain are linked together via a disulfide bond. The Fab of the present disclosure can be obtained by treating a monoclonal antibody of the present invention (which specifically recognizes human Abeta and binds to the extracellular region or its three-dimensional structure) with papain. In addition, a Fab can be obtained by incorporating DNA encoding an antibody Fab into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryote or eukaryote to express the Fab.

As used herein, F(ab') ₂ is an antibody fragment having a molecular weight of about 100,000 and having antigen-binding activity and containing two Fab regions that are linked in a hinge position, it can be obtained as a result of pepsin cleavage of two disulfide bonds at the bottom of the hinge region of IgG. The F(ab') ₂ of the present disclosure can be obtained by pepsinizing the monoclonal antibody of the present invention (which specifically recognizes human Abeta and binds to the extracellular region or its three-dimensional structure). In addition, F(ab') ₂ can be obtained by linking Fab', described below, thioether bond or disulfide bond.

As used herein, a Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity. Fab' is obtained by cleavage of the disulfide bond in the hinge region of the above F(ab') ₂ . The Fab' of the present disclosure can be obtained by treating F(ab') ₂ of the present disclosure (which specifically recognizes Abeta and binds to the extracellular region or its three-dimensional structure) with a reducing agent such as dithiothreitol. Furthermore, a Fab' can be obtained by incorporating the DNA encoding the Fab' of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryote or eukaryote to express the Fab'.

As used herein, the term "single chain antibody", "single chain Fv", or "scFv" refers to a molecule containing an antibody heavy chain variable domain (or region; VH) coupled to an antibody light chain variable domain. (or region; VL) with a linker. Such scFv molecules have the general structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. A suitable linker in the prior art consists of the repeat amino acid sequence GGGGS or a variant thereof, for example a variant with 1-4 repeats (Holligeret al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. The scFv of the present disclosure can be obtained through the following steps: obtaining a cDNA encoding VH and VL of a monoclonal antibody of the present disclosure that specifically recognizes human Abeta and binds to an extracellular region or its three-dimensional structure, constructing a DNA encoding scFv; incorporating the DNA into a prokaryotic expression vector or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express said scFv.

As used herein, a diabody is an antibody fragment wherein the scFv is dimerized, and it is an antibody fragment having bivalent antigen-binding activity. With bivalent antigen-binding activity, the two antigens may be the same or different. The diabody of the present disclosure can be obtained through the following steps: obtaining cDNAs encoding VH and VL of a monoclonal antibody of the present disclosure that specifically recognizes human Abeta and binds to the extracellular region or its three-dimensional structure; constructing a DNA encoding scFv such that the length of the linker peptide is 8 amino acid residues or less, incorporating the DNA into a prokaryotic expression vector or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the diabody.

As used herein, dsFv is produced by replacing one amino acid residue in each of VH and VL with a cysteine residue, and then linking the substituted polypeptides via a disulfide bond between these two cysteine residues. Amino acid residues to be replaced by a cysteine residue can be selected based on the prediction of the three-dimensional structure of the antibody according to known methods (Protein Engineering, 7, 697 (1994)). dsFv of the present disclosure can be obtained through the following steps: obtaining cDNAs encoding VH and VL of the monoclonal antibody of the present disclosure, which specifically recognizes human Abeta and binds to the extracellular region or its three-dimensional structure; constructing a DNA encoding a dsFv, incorporating the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the dsFv.

As used herein, a CDR-containing peptide is constructed from one or more CDR, VH, or VL regions. Peptides containing multiple CDRs can be linked directly or through a suitable peptide linker. The CDR-containing peptide of the present disclosure can be produced by the following steps: constructing a DNA encoding the VH and VL of a monoclonal antibody of the present disclosure that specifically recognizes human Abeta and binds to the extracellular region or its three-dimensional structure, incorporating the DNA into a prokaryotic expression vector, or a eukaryotic expression vector, and then introducing this expression vector into a prokaryote or eukaryote to express the peptide. The CDR-containing peptide can also be prepared by a chemical synthesis method such as the Fmoc (9-fluorenylmethoxycarbonyl) method or the tBoc (tert-Butoxycarbonyl) method.

As used herein, the term "CDR" refers to one of the six hypervariable regions found in the variable domain of an antibody that primarily facilitate antigen binding. One of the most widely used definitions for 6 CDR data was proposed by Kabat E.A. et al, ((1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat CDR definition applies to light chain variable domain KCDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) and to CDR2 and CDR3 of the variable light chain. heavy chain domain (CDR H2, CDR H3 or H2, H3).

As used herein, the term "antibody framework region" refers to the portion of a variable domain, or VL or VH, that serves as the backbone for the antigen-binding loops (CDRs) of that variable domain. Essentially, it is a variable domain without a CDR.

As used herein, the term "epitope" or "antigenic determinant" or "antigen epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on an Abeta molecule). Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a single tertiary conformation. (See, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, ed. G. E. Morris (1996)).

As used herein, the term "specifically binds to", "selectively binds to", "selectively binds to", or "specifically binds to" refers to the binding of an antibody to a given epitope on an antigen. Typically, an antibody will bind with an affinity (KD) of less than about 10 ^-7 M, such as less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M, or even less.

As used herein, the term "KD" or "Kd" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, an antibody of the present disclosure will bind to human Abeta with a dissociation equilibrium constant (KD) of less than about 10 ^-7 M, such as less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M, or even less, such as determined by surface plasmon resonance (SPR) technology on a Biacore T200 instrument.

As used herein, the terms "competitive binding" and "competitively binds to" refer to an antibody that recognizes and binds to the same epitope (also called an antigenic determinant) or part of the same epitope on human Abeta that and monoclonal antibodies of the present disclosure. An antibody that binds to the same epitope as the monoclonal antibodies of the present disclosure refers to an antibody that recognizes and binds to the human Abeta amino acid sequence that the monoclonal antibodies of the present disclosure recognize.

When the term "competition" is used in the context of antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) that compete for the same epitope, it means that competition occurs between antigen-binding proteins, which is determined by assays in which the antigen-binding protein, the target to be tested (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen-binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., PD-L1 antigen or fragment thereof). Many types of competitive binding assays are available to determine if one antigen binding protein competes with another. These analyzes are, for example, solid-phase direct or indirect radioimmunoassay (RIA - from the English radioimmunoassay), solid-phase direct or indirect enzyme immunoassay (EIA - from the English enzyme immunoassay), competitive "sandwich" analysis (see, for example, Stahli et al, 1983, Methods in Enzymology 9: 242-253); solid phase direct EIA with biotin-avidin (see e.g. Kirkland et al, 1986, J. Immunol. 137: 3614-3619), solid phase direct labeling assay, solid phase direct labeled sandwich assay (see eg Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct RIA based on labeling with label I ¹²⁵ (see, for example, Morel et al, 1988, Molec. Immunol. 25: 7-15); solid phase direct EIA with biotin-avidin (see, for example, Cheung, et al, 1990, Virology 176: 546-552); and label-based direct RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves the use of a purified antigen capable of binding to a solid surface or cell loaded with both the unlabeled test antigen-binding protein and the labeled reference antigen-binding protein. Competitive inhibition is determined by measuring the amount of label bound to a solid surface or to a cell in the presence of the antigen-binding protein being tested. Typically, the antigen binding protein being tested is in excess. Antigen-binding proteins identified by competitive analysis (competing with an antigen-binding protein) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to an epitope that is sufficiently close to the epitope to which the reference antigen-binding protein binds, where the two epitopes spatially interfere with each other to prevent binding. Additional details regarding methods for determining competitive binding are provided herein in the Examples section. Typically, when the competing antigen binding protein is in excess, it will inhibit (eg, reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70 %, 70-75% or 75% or even more specific binding of the reference antigen-binding protein to a common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97% or 97%, or even more.

The term "nucleic acid molecule", as used herein, refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single or double stranded, but is preferably double stranded DNA. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence.

The term "vector", as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In yet another embodiment, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. The vectors disclosed herein are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial replicator and mammalian episomal vectors), or can be integrated into the genome of a host cell when introduced into the host cell. and thus replicate along with the host genome (eg, non-episomal mammalian vectors).

The term "host cell", as used herein, refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria that are susceptible to transformation include members of the Enterobacteriaceae such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.

Methods for preparing and purifying antibodies and antigen-binding fragments are well known in the art, for example, see A Laboratory Manual for Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-8 and 15. For example, mice can be immunized with human Abeta or fragments thereof, and the resulting antibodies can then be annealed, purified and sequenced for amino acid sequences using conventional methods well known in the art. Antigen binding fragments can also be obtained by conventional methods. The antibodies or antigen-binding fragments of the present disclosure are designed to contain one or more framework regions and CDRs derived from a non-human antibody. Human germline FR sequences can be obtained from ImMunoGeneTics (IMGT) via their website http://imgt.cines.fr or from The Immunoglobulin Facts Book, 2001, ISBN 012441351, by aligning against sequences from the Antibody Variable Region Gene Database human germline and use of the MOE software.

The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using known methods. For example, cDNA sequences encoding the heavy chain and light chain can be cloned and constructed into a GS expression vector. Vectors expressing the recombinant immunoglobulin can then be stably transfected into CHO cells. As a more preferred method, well known in the art, mammalian expression systems will result in glycosylation, typically at highly conserved N-terminal sites in the Fc region. Received stable clones expressing an antibody that specifically binds to human TIM-3. Positive clones can be expanded in serum-free culture media in bioreactors to produce antibodies. The culture medium into which the antibody has been secreted can be purified by conventional techniques. For example, purification can be carried out on a Sepharose FF protein A or G column that has been modified with a buffer. The non-specific binding components are washed out. The bound antibody is eluted with a pH gradient and the antibody fragments are detected by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and then pooled. Antibodies can be filtered and concentrated using conventional techniques. Soluble aggregates and multimers can be effectively removed by conventional techniques such as gel filtration or ion exchange. The resulting product is then immediately frozen, for example at -70°C, or it can be lyophilized.

As used herein, the term "mutation" includes, but is not limited to, "back mutation", "conservative modification", or "conservative substitution". As used herein, the term "conservative modification" or "conservative substitution" refers to substitutions of amino acids in a protein with other amino acids that have similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, conformation, and rigidity). backbone, etc.), so that these changes can often occur without changing the biological activity of the protein. It will be recognized by those skilled in the art that, in general, a single amino acid substitution in the interchangeable regions of a polypeptide does not significantly alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p .224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to impair biological activity.

The term "mutant sequence" referred to in the present disclosure refers to a nucleotide sequence and an amino acid sequence having a different sequence identity, expressed as a percentage, with the sequences of the present disclosure after the nucleotide sequence and amino acid sequence of the present disclosure are modified by an appropriate substitution, insertion, or deletion. . The sequence identity described in this disclosure may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% . Sequence comparison and determination of percent identity between two sequences can be performed using the default BLASTN/BLASTP algorithm available from the National Center for Biotechnology Institute website.

The term "homology" or "identity" or "correspondence", as used herein, refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences. When a position in both of the two sequences to be compared is occupied by the same monomeric subunit - base or amino acid - for example, if a position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology of two sequences is a function of the number of matching or homologous positions that the two sequences share, divided by the number of positions compared and then multiplied by 100. For example, in an optimal sequence alignment, if 6 out of 10 positions in two sequences are the same or homologous, then these two sequences have 60% homology. If 95 out of 100 positions in two sequences match or are homologous, then the two sequences have 95% homology. In general, a comparison is made when two sequences are aligned to obtain the maximum percent homology.

The term "exogenous" as used in this disclosure refers to substances produced outside organisms, cells, or the human body, as the case may be. The term "endogenous" refers to substances produced in cells, organisms or the human body, as the case may be.

As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably, and all such designations include their progeny. Thus, the words "transformant" and "transformed cell" include the primary test cells and cultures derived from them, without regard to the number of transfers. It should also be understood that all progeny may not be exactly identical in DNA content due to targeted or random mutations. Included are progeny with mutations that have the same function or have the same biological activity as the function or biological activity in the original transformed cells. When distinct designations are intended, this will be evident from the context.

As used herein, the phrase "polymerase chain reaction" or "PCR" refers to a process or procedure in which small amounts of a particular nucleic acid fragment, RNA and/or DNA, are amplified as described, for example, in US Pat. No. 4,683,195. Typically there is a need for sequence information at the ends or outside of the region of interest so that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to the reverse strands of the template to be amplified. The 5'-terminal nucleotides of these two primers may coincide with the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA and cDNA transcribed from total cell RNA, bacteriophage or plasmid sequences, and so on. Generally see Mullis et al. (1987) Cold Spring Harbor Symp.Ouant.Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). The PCR assay used in the present disclosure is considered to be one, but not the only, example of a method for amplifying a nucleic acid test sample based on a polymerase reaction. The method includes using known nucleic acid sequences as primers and a nucleic acid polymerase to amplify or generate a particular nucleic acid fragment.

The term "pharmaceutical composition", as used herein, refers to a mixture containing one or more compounds according to the present disclosure or its (their) physiologically/pharmaceutically acceptable salt or prodrug and other chemical components, such as physiologically /pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to the body, to facilitate absorption of the active substance and thus to exert a biological effect.

The term "treat", as used herein, means to administer orally or topically a therapeutic agent, such as a composition containing any of the binding compounds of the present disclosure, to a patient who has one or more of the symptoms of a disease for whom this agent has a known therapeutic activity. Typically, the agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, or to cause regression or inhibit the progression of such symptom(s) to any clinically measurable degree. The amount of a therapeutic agent that is effective in alleviating any particular symptom of a disease (also referred to as a "therapeutically effective amount") may vary depending on various factors such as the course of the disease, the age and weight of the patient, and the ability of the drug to elicit the desired response. at the patient. Whether a symptom of a disease has been alleviated can be assessed by any clinical measurement commonly used by physicians or other qualified healthcare professionals to assess the severity or progression status of that symptom. While an embodiment of the present invention (e.g., method of treatment or product of manufacture) may not be effective in alleviating the target symptom(s) of the disease in each patient, it should alleviate the target symptom(s) of the disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as Student's t-test, chi-square test, Mann and Whitney U-test, Kruskal-Wallis (H-test), Jonkheer-Terpstra test, and Wilcoxon test .

The term "effective amount", as used herein, encompasses an amount sufficient to alleviate or prevent a symptom or sign of a medical condition. An effective amount also means an amount sufficient to enable or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing protocol that avoids significant side effects or toxic effects.

The term "administration" or "treatment", as used herein, when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the administration of an exogenous pharmaceutical, therapeutic, diagnostic means or compositions in contact with an animal, human, subject, cell, tissue, organ or biological fluid. The term "administration" or "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Processing a cell encompasses bringing a reagent into contact with a cell, as well as bringing the reagent into contact with a fluid, where the fluid is in contact with the cell. The term "administration" or "treatment" also means in vitro or ex vivo treatments of, for example, a cell with a reagent, diagnostic agent, binding composition, or other cell. The term "treatment", when applied to a human subject, a veterinary subject, or a research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

As used herein, the term "disease or disorder caused by Abeta" refers to a disease or disorder caused by or associated with β-amyloid, including, but not limited to, diseases and disorders caused by the presence or activity of β -amyloid composed of Abeta monomers, or protofibrils or polymers, or any combination thereof, e.g., a neurodegenerative disease (e.g., Alzheimer's disease, mild cognitive impairment, frontotemporal dementia, Lewy body dementia, Parkinson's disease, Pick's disease, Bevac's disease, etc.). etc.), various diseases of the eye caused by β-amyloid deposition (eg, macular degeneration, drusen-associated optic neuropathy, glaucoma, cataracts, etc.).

As used herein, "neurodegenerative diseases" include the following: Alzheimer's disease, mild cognitive impairment, frontotemporal dementia, Lewy body dementia, Parkinson's disease, Pick's disease, Bewak's disease, congophilic amyloid angiopathy, cerebral amyloid angiopathy , Down syndrome, multi-infarct dementia, Huntington's disease, Creutzfeldt-Jakob disease, AIDS dementia syndrome, depression, anxiety disorder, phobia, Bell's palsy, epilepsy, encephalitis, multiple sclerosis, neuromuscular disorder, neuro-oncological disease, brain tumor, neurovascular stroke disorder, neuroimmunological disease, neuropathic otological disease, traumatic injury to the nervous system, including spinal cord injury, pain, including neuropathic pain, childhood neuro- and neuropsychiatric disorder, sleep disturbance, Tourette's syndrome, cognitive lung disease, vascular dementia, multi-infarct dementia, cystic fibrosis, Gaucher's disease and other dyskinesia and diseases of the central nervous system, but are not limited to.

Detailed description of the invention

The following examples are offered to further describe the present disclosure, but are not intended to limit the scope of the disclosure. Experimental methods for which no specific conditions are specified in the examples of this disclosure are generally carried out according to conventional conditions, such as those of Sambrook et al., Antibodies, Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; or in accordance with the conditions recommended by the manufacturer. Reagents for which sources are not specifically indicated are commercially available reagents.

Example 1 Obtaining the Abeta Antigen

The Abeta antigens used in the Examples and Assay Examples of this disclosure were synthesized using GL Biochem, GenScript or Sigma and then generated in vitro (see Table 1 for more details). Abeta antigens for use in immunization were Aβ 1-42 and Aβ 1-40 KLH (Keyhole Limpet Hemocyanin) protofibrils; Abeta antigens for use in screening were Aβ1-40-BSA fibrils (bovine serum albumin), Aβ1-40 DMSO suspension, Aβ1-42-biotin DMSO suspension , Aβ1-42 protofibrils and Aβ1-42 fibrils; Abeta antigens for use in detection were Aβ1-42 monomer and Aβ1-42 fibrils.

The amino acid sequence of Aβ 1-40 is shown in (SEQ ID NO 1): DAEFRHDSGYEVHQKLVFFAEDVGSNKGAIIGLMVGGVV;

The amino acid sequence of Aβ 1-42 is shown in (SEQ ID NO 2): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA.

Aβ1-40 KLH and Aβ1-40-BSA refer to the synthesized Aβ1-40 polypeptide fused to KLH (keyhole limpet hemocyanin) and BSA (bovine serum albumin), respectively, where the Aβ1-40 used was included with the amino acid Cys at the N-terminus for ease of linking and detection;

Aβ1-42 protofibrils refer to the Aβ1-42 polypeptide obtained as protofibrils in vitro.

Aβ1-40-BSA fibrils refers to an Aβ1-42 polypeptide coupled to BSA, obtained as fibrils in vitro.

Aβ1-40 DMSO suspension refers to a clear solution prepared by directly dissolving the Aβ1-42 polypeptide in DMSO (dimethyl sulfoxide).

Suspension Aβ1-42-Biotin DMSO refers to a clear solution prepared by directly dissolving the Aβ1-42 polypeptide labeled with biotin in DMSO (dimethyl sulfoxide).

Aβ1-42 fibrils refer to the Aβ1-42 polypeptide obtained as fibrils in vitro.

The Aβ1-42 monomer refers to the Aβ1-42 monomer.

Example 2 Preparation of anti-human Abeta hybridoma monoclonal antibody

1. Immunization

Monoclonal antibodies against human Abeta were obtained by immunization of mice. SJL white mice, female, 6-8 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001) were used for the experiment. Feeding environment: SPF level (from the English specific pathogen free - free from specific pathogenic microflora). After acquisition, the mice were kept in the laboratory for 1 week, with a 12/12 hour light/dark cycle, at 20-25°C; humidity 40-60%. Mice were then immunized according to the following schemes. Antigens for immunization were Aβ1-40-KLH and Aβ1-42 protofibrils.

Immunization Schedule 1: The adjuvant used for immunization was QuickAntibody-Mouse3W (Beijing Kangbiquan Biotechnology Co., Ltd. Cat. No. KX0210042). The ratio of antigen and adjuvant (QuickAntibody-Mouse3W) was 1:1, 10 μg/mouse/time period. Antigen and adjuvant were mixed thoroughly and used for inoculation on days 0, 7, 14, 21, 28, 35 and 42. On day 0 mice were injected intramuscularly (IM) in the hindpaw with 10 μg/mouse of the mixed antigen. The first immunization was repeated on days 7, 14, 21, 28, 35 and 42, and mice were similarly injected intramuscularly (IM) in the hindpaw with 10 μg/mouse of the mixed antigen. Blood samples were collected on days 19, 40 and 55, and the antibody titer in mouse serum was determined by ELISA. After the seventh to eighth immunization, mice with a high serum antibody titer tending to plateau were selected for splenocyte fusion. Three days prior to splenocyte fusion, an antigen solution prepared with saline was injected intraperitoneally (ip), 20 μg/mouse, for booster immunization.

Immunization Schedule 2: The adjuvant used for immunization was QuickAntibody-Mouse5W (Beijing Kangbiquan Biotechnology Co., Ltd. Cat. No. KX021004). The ratio of antigen and adjuvant (QuickAntibody-MouseSW) was 1:1, 25 μg/mouse/time period (for the first immunization) and 25 μg/mouse/time period (for booster immunization). Antigen and adjuvant were mixed thoroughly and used for inoculation on days 0, 14, 28, 42 and 56. On day 0 mice were injected intramuscularly (IM) in the hindpaw with 25 μg/mouse of the mixed antigen. The first immunization was repeated on days 14, 28, 42 and 56, and mice were similarly injected intramuscularly (IM) in the hindpaw with 25 μg/mouse of the mixed antigen. Blood samples were taken on days 20 and 49 and the mouse serum antibody titer was determined by ELISA. After the fifth immunization, mice with a high serum antibody titer tending to plateau were selected for splenocyte fusion. Three days prior to splenocyte fusion, an antigen solution prepared with saline was injected intraperitoneally (ip), 50 μg/mouse, for booster immunization.

2. Splenocyte fusion

Hybridoma cells were generated by fusion of spleen lymphocytes with Sp2/0 myeloma cells (ATCC® CRL-8287™) using an optimized PEG (polyethylene glycol) mediated fusion method. Merged hybridoma cells were resuspended in a complete medium (DM EM medium (Dulbecco modified Eagle's medium - Dulbecco's modified Eagle's medium) containing 20% FBS (Fetal Bovine Serum - fetal calf serum), 1 * HAT (from English hypoxanthine-aminopterinethymidine - hypoxanthine aminopterinethymidine), 1×ORI) with a density of 0.5-1*10 ⁶ /ml, seeded in a 96-well plate in the amount of 100 μl/well, incubated at 37°C and 5% CO ₂ for 3-4 days, 100 μl/well of complete HAT medium was added and cultured for 3-4 days until needle-like clones were formed. The supernatant was removed, 200 μl/well of complete HT medium (RPMI-1640 medium containing 20% FBS, 1*HAT and 1*OP1) was added, incubated at 37°C, 5% CO ₂ for 3 days and then subjected to ELISA detection .

3. Screening for hybridoma cells

10-11 days after fusion, according to the density of the growing cells, an ELISA binding assay (also known as enzyme-linked immunosorbent assay) was performed on the clone culture supertantant with different forms of the Aβ polypeptide for pre-screening to classify the subtypes of the resulting hybridoma clones. Aβ1-40-BSA fibrils, Aβ1-42-biotin DMSO suspension and Aβ 1-40 DMSO suspension were used to screen CA01-40-KLH hybridoma cells as an immunogen; and Aβ1-42 protofibrils, Aβ1-42 fibrils, and Aβ1-42-biotin DMSO suspension were used to screen hybridoma cells with Aβ1-42 protofibrils as an immunogen. In the case of positive wells, the medium was changed and placed in a 24-well plate according to cell density. The cell lines transferred to the 24-well plate were tested again and then subcloned for the first time. Some of the positive cells screened in the first subcloning were preserved and used for the second subcloning. Some of the positive cells screened in the second subcloning were preserved and used for protein expression. Hybridoma cells capable of specifically binding to the Aβ polypeptide have been obtained from multiple fusions. After thioflavin (also referred to as ThT) fluorescence assay, neuronal protection assay, and macrophage phagocytosis assay, hybridoma clone mAb-2401, mAb-3601, mAb-5101 and mAb-9001 was obtained and used to generate antibodies with serum-free cell culture. The resulting antibodies were purified according to the purification example, and the purified antibodies were used in the analysis examples.

4. Purification of hybridoma antibodies and recombinant antibodies

Cell-expressed supernatant samples were centrifuged at high speed to remove impurities, and the hybridoma-expressed supernatant was purified by a Protein G column, and the recombinant antibody supernatant was purified by a Protein A column. The column was washed with PBS (pH 7.4 ) until the measured value A280 drops to the original level. Target proteins were eluted with 100 mM acetic acid, pH 3.0 and neutralized with 1 M Tris-HCl, pH 8.0. The eluted samples were suitably concentrated and then further purified with a Superdex200 (GE) gel chromatography column pre-equilibrated with PBS to remove aggregates. Monomer peaks were collected and aliquoted for use.

5. Sequencing of positive hybridoma clones

Methods for cloning sequences from a positive hybridoma were as follows. Hybridoma cells were collected in the logarithmic growth phase. RNA was isolated with Trizol (Invitrogen cat. no. 15596-013) according to kit instructions and reverse transcribed with the PrirneScript™ reverse transcription kit (Takara cat. no. 2680A). The resulting cDNAs were amplified by PCR using a mouse Ig primer set (Novagen, TB326 Rev. B 0503) and sequenced in-house. The amino acid sequences of the mouse antibody heavy chain variable region (VH) and light chain variable region (VL) of mAb-2401, mAb-3601, mAb-5101 and mAb-9001 hybridoma clones were determined as follows:

Note: The amino acid sequences of the variable regions of the above antibodies are shown as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Letters in italics indicate FR sequences and underlined letters indicate CDR sequences.

The amino acid sequences of the CDR regions of mAb-2401, mAb-3601, mAb-5101 and mAb-9001 are shown in Table 2.

Example 3 Humanization of anti-human Abeta hybridoma monoclonal antibodies

The canonical structure was determined according to the light chain variable region sequence and the heavy chain variable region sequence of the mouse antibody. Among human germline sequences with the same structure, the most mouse-like non-CDR regions (framework regions) were selected as templates for humanization. Then, the mouse antibody CDR regions were grafted onto selected humanization templates to replace the human CDR regions. Back mutations or point mutations were made on some sequences of the FR regions, if necessary. The variable regions are then combined with human constant region(s) (such as human IgG1 constant region) to generate humanized anti-Abeta antibodies.

1. Humanization of mAb-2401

The mAb-2401 heavy chain humanization templates were IGHV1-24*01 and hjh6.3. Namely, the FR1, FR2 and FR3 regions were selected from the human germline heavy chain IGHV1-24, and the JH6 hjh6.3 region was selected as the FR4 region. Light chain templates were IGKV1-27*01 and hjk4.1. Namely, FR1, FR2 and FR3 were selected from the human germline light chain IGKV1-27, and the JK4 region of hjk4.1 was selected as the FR4 region.

The amino acid sequence of the human germline heavy chain template (IGHV1-24*01) is shown in (SEQ ID NO 28):

The amino acid sequence of the human germline light chain template (IGKV 1-27*01) is shown in (SEQ ID NO 29):

The humanized variable regions of the heavy and light chains were as follows:

HAB-2401 with VH grafted (SEQ ID NO 66):

HAB-2401 with VL grafted (SEQ ID NO 67):

Mouse antibody CDR regions were grafted onto a selected humanization template to replace the human CDR regions of the template, and backmutations were introduced into the heavy chain at positions 1, 72, and 98 (natural sequence numbering corresponding to position 1, 71, and 94, respectively, according to Kabat numbering). ) (Q1E, E72A and T98R; Q1E, E71A, T94R according to Kabat numbering), and then combined with the constant region of human IgG 1 to obtain a humanized HAB-2401 antibody.

The amino acid sequence of the heavy chain of HAB-2401 is shown in (SEQ ID NO 30):

The amino acid sequence of the HAB-2401 light chain is shown in (SEQ ID NO 31):

Note: In the above amino acid sequences of the HAB-2401 light and heavy chains, the italicized letters indicate the FR sequence, the underlined letters indicate the CDR sequence, the double underlined letters indicate the region constant sequence.

2. Humanization of mAb-3601

The mAb-3601 heavy chain humanization templates were IGHV3-30*01 and hjh6.3. Namely, the FR1, FR2 and FR3 regions from the human germline heavy chain IGHV3-30 were selected, and the JH6 hjh6.3 region was selected as the FR4 region. Light chain templates were IGKV1-39*01/hjk4.1. Namely, FR1, FR2 and FR3 were selected from the human germline light chain IGKV1-39, and the JK4 region of hjk4.1 was selected as the FR4 region.

The amino acid sequence of the human germline heavy chain template (IGHV3-30*01) is shown in (SEQ ID NO 32):

The amino acid sequence of the human germline light chain template (IGKV1-39*01) is shown in (SEQ ID NO 33):

The humanized variable regions of the heavy and light chains were as follows:

HAB-3601 VH grafted (SEQ ID NO 68):

HAB-3601 with VL graft (SEQ ID NO 69):

Mouse antibody CDR regions were grafted onto a selected humanization template with replacement of human CDR regions from the template, backmutations were introduced on the heavy chain at positions 28 and 86 (natural sequence numbering corresponding to position 28 and 82B, respectively, according to Kabat numbering) (T28A and S86T; T28A and S82B T according to Kabat numbering), a point mutation was introduced on the heavy chain at position 58 (natural sequence numbering) (D58N) to eliminate possible isomerization of aspartic acid and then combined with a human IgG1 constant region to obtain a humanized HAB-3601 antibody.

The amino acid sequence of the heavy chain of HAB-3601 is shown in (SEQ ID NO 34):

The amino acid sequence of the HAB-3601 light chain is shown in (SEQ ID NO 35):

Note: In the amino acid sequences of the HAB-3601 light and heavy chains above, italicized letters indicate the FR sequence, underlined letters indicate the CDR sequence, and double underlined letters indicate the constant region sequence.

3. Humanization of mAb-5101

The mAb-5101 heavy chain humanization templates were IGHV2-70D*04 and hjh6.1. Namely, FR1, FR2 and FR3 were selected from the human germline heavy chain IgHV2-70D, and the JH6 region of hjh6.1 was selected as the FR4 region. Light chain templates were IGKV2-40*01 and hjk4.1. Namely, the FR1, FR2 and FR3 regions were selected from the human germline light chain IGKV2-40, and the JK4 region of hjk4.1 was selected as the FR4 region.

The amino acid sequence of the human germline heavy chain template (IGHV2-70D*04) is shown in (SEQ ID NO 36):

The amino acid sequence of the human germline light chain template (IGKV2-40*01) is shown in (SEQ ID NO 37):

The humanized variable regions were as follows: HAB-5101 grafted with VH (SEQ ID NO 70):

HAB-5101 with VL graft (SEQ ID NO 71):

Mouse antibody CDR regions were grafted onto a selected humanization template with replacement of human CDR regions from the template, backmutations were made on the light chain at position 2 and 50 (natural sequence numbering corresponding to position 2 and 45, respectively, according to Kabat numbering) (I2V and Q50K; I2V and Q45K according to Kabat numbering), a point mutation was introduced on the light chain at position 94 (natural sequence numbering) (C94S) to exclude a possible mismatch involving a disulfide bond and then combined with a human IgG1 constant region to obtain a humanized HAB antibody -5101.

The amino acid sequence of the heavy chain of HAB-5101 is shown in (SEQ ID NO 38):

The amino acid sequence of the HAB-5101 light chain is shown in (SEQ ID NO 39):

Note: In the amino acid sequences of the HAB-5101 light and heavy chains above, italicized letters indicate the FR sequence, underlined letters indicate the CDR sequence, and double underlined letters indicate the constant region sequence.

4. Humanization of mAb-9001

The mAb-9001 heavy chain humanization templates were IGHV2-70D*04 and hjh6.1. Namely, FR1, FR2 and FR3 were selected from the human germline heavy chain IGHV2-70, and the JH6 region of hjh6.1 was selected as the FR4 region. Light chain templates were IGKV2-40*01 and hjk2.1. Namely, FR1, FR2 and FR3 were selected from the human germline light chain IGKV2-40, and the JK2 region of hjk2.1 was selected as the FR4 region.

The amino acid sequence of the human germline heavy chain template (IGHV2-70D*04) is shown in (SEQ ID NO 36):

The amino acid sequence of the human germline light chain template (IGKV2-4CT01) is shown in (SEQ ID NO 37):

The humanized variable regions were as follows: HAB-9001 grafted with VH (SEQ ID NO 72):

HAB-9001 with VL graft (SEQ ID NO 73):

Mouse antibody CDR regions were grafted onto a selected humanization template with replacement of human CDR regions from the template, backmutation was introduced on the light chain at position 2 (numbering of natural sequences corresponding to position 2 according to Kabat numbering) (I2V), point mutation was introduced on the heavy chains at position 58 (numbering of natural sequences) (D58N) to exclude possible isomerization of aspartic acid, and then combined with a human IgG1 constant region to obtain a humanized HAB-9001 antibody.

The amino acid sequence of the heavy chain of HAB-9001 is shown in (SEQ ID NO 40):

The amino acid sequence of the HAB-9001 light chain is shown in (SEQ ID NO 41):

Note: In the above amino acid sequences of the HAB-9001 light and heavy chains, italicized letters indicate the FR sequence, underlined letters indicate the CDR sequence, and double underlined letters indicate the constant region sequence.

In addition, with respect to the antibody constant region sequences HAB-2401, HAB-3601, HAB-5101 and HAB-9001, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO 42, the amino acid sequence of the light chain constant region is shown in SEQ ID NO 43 , variable region amino acid sequences, and full-length amino acid sequences are shown in the following table:

Example 4 Production of Humanized Antibodies 1. Molecular Cloning of Humanized Antibodies Codon optimization was performed against engineered humanized antibody sequences to obtain human codon-preferred gene coding sequences. The VH/VK gene fragment of each antibody was constructed with the designed PCR primers and then introduced into the pHr expression vector (carrying the signal peptide and the constant region gene fragment (CH1-FC/CL)) by homologous recombination to construct a plasmid expressing the full-length humanized VH- antibody CH1-FC-pHr/VK-CL-pHr. The correctness of the clones was checked by sequencing, where, in the case of HAB-2401, the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO 54, the nucleotide sequence encoding the light chain is shown in SEQ ID NO 55; in the case of HAB-3601, the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO 56, the nucleotide sequence encoding the light chain is shown in SEQ ID NO 57; in the case of HAB-5101, the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO 58, the nucleotide sequence encoding the light chain is shown in SEQ ID NO 59; and in the case of HAB-9001, the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO 60, the nucleotide sequence encoding the light chain is shown in SEQ ID NO 61.

2. Expression and purification of humanized antibodies

HEK293E cells were transfected with plasmids expressing the heavy and light chains of the antibody, respectively, in a ratio of 1:1.5. Six days after transfection, the expression supernatant was collected, centrifuged at high speed to remove contaminants, and purified via a protein A column. The column was washed with PBS until the A280 value dropped to baseline. Target proteins were eluted with 100 mM acetic acid, pH 3.0 and neutralized with 1 M Tris-HCl, pH 8.0. The eluted samples were suitably concentrated and then further purified with a Superdex200 (GE) gel chromatography column pre-equilibrated with PBS to remove aggregates. Monomer peaks were collected and al and quoted for use.

The efficacy and efficacy of the present disclosure was tested using the following biochemical assays or in vivo pharmacological assays:

Assay Example 1 Affinity of Anti-Abeta Antibodies Detected by BIAcore Assay

The affinity of the antibody to Aβ1 fibrils was determined by the amino group binding method. Aβ1-42 fibrils were bound to a CM5 (200 RU) biosensor chip, and then the antibody molecules were allowed to pass through the surface of this chip. Interaction signals were detected in real time using a Biacore T200 instrument to generate a binding and dissociation curve. At the end of each experimental cycle, after completion of dissociation, the biosensor chip was washed and regenerated with 10 mM Glycine-HCI (pH 1.5). An antibody positive control, Aducanumab, was prepared according to the WHO (World Health Organization) Aducanumab Sequence Information (See WHO Drug Information, Vol. 28, No. 3, 2014) and the production method is described in Example 4 of this disclosure. .

The affinity of the antibody for the Aβ1-42 monomer was determined by means of a capture antibody on a chip. IgG was captured by affinity on the Protein A biosensor chip, and then the Aβ1-42 monomer antigen was allowed to pass through the surface of the chip. Interaction signals were detected in real time using a Biacore T200 instrument to obtain a binding and dissociation curve. At the end of each experimental cycle, after completion of dissociation, the biosensor chip was washed and regenerated with 10 mM Glycine-HCI (pH 1.5).

The experimental results are shown in Table 4. HAB-2401, HAB-5101, HAB-3601 and HAB-9001 have an affinity for Aβ1-42 fibrils ranging from 10 ^-8 to 10 ^-10 M, of which HAB-9001 has the strongest affinity, which is 2 orders of magnitude stronger than the affinity of aducanumab. HAV-2401, HAV-5101, HAV-3601 and HAV-9001 have an affinity for the Aβ1-42 monomer ranging from 10 ^-7 to 10 ^-8 M, of which HAV-9001 has the strongest affinity, which is 2 order of magnitude stronger than the affinity of aducanumab. All of the four antibodies analyzed have an affinity that is stronger than that of the positive control antibody, Aducanumab.

Assay Example 2: Effect of anti-Abeta antibodies on blocking Aβ1-42 monomer aggregation into Aβ1-42 fibrils

The effect of anti-Abeta antibodies on the inhibition of Aβ1-42 monomer aggregation into Aβ1-42 fibrils was determined by ThT-based assay. ThT (Thioflavin T, thioflavin) is a type of fluorescent dye and is commonly used for the in vitro identification of Aβ fibrils because fluorescence signals at an excitation wavelength of 450 nm and an emission wavelength of 482 nm will be greatly enhanced when it is combined with a β-sheet rich protein (such as the deposition of amyloid Ap). Specific experimental ThT-based assay procedures were specified as follows. 0.5mg of human Aβ1-42 (GenScript Cat # RP10017) was added to 250 µl of 10 mM NaOH, dissolved completely, and then added to an equal volume of pre-chilled 2xPBS to neutralize. A 1 mg/ml solution of Aβ1-42 monomer was prepared and diluted with ddH ₂ O to 0.25 mg/ml. Aβ1-42 monomer (10 µl/well, 0.25 mg/ml) was mixed with 20 µM ThT (sigma, cat. no. T3516) and added to a black 384-well plate (Corning, cat. no. 4514), and the well without the addition of Aβ 1-42 monomer served as a negative control. Then, gradient diluted antibodies (starting at 1 mg/ml, 2-fold gradient dilution, 10 µl/well) were added to the above mixture, incubated at 37°C for 24 h. Fluorescence signals were read at an excitation wavelength of 440 nm and wavelength of 485 nm using the FlexStation3 microplate reader (Molecular Devices).

The experimental results are shown in Fig. 1. All of HAB-2401, HAB-5101 and HAB-9001 can effectively inhibit the aggregation of Aβ1-42 monomers into Aβ1-42 fibrils. Whereas NAV-3601 and Aducanumab do not perform such a function.

Assay Example 3 Anti-Abeta Antibody Protection on Primary Rat Neurons

Cerebral cortex was excised from SD fetal rats from gestational age 16-18 days, digested with trypsin (Gibco cat. no. 25200-072), pipetted and filtered into a single cell suspension, centrifuged and counted. Cells were diluted with DM EM (Gibco, cat. no. 10564-029) containing 10% FBS (Gibco, cat. no. 10099-141) to 1E4 per well (1×10 ⁴ /well) and plated in a 96-well plate ( Corning, cat. No. 3903), coated with PDL (from the English. Poly-D-lysine - poly-D-lysine, polylysine) (Sigma, cat. No. P0899). After overnight incubation in an incubator (37°C, 5% CO ₂ ), medium was replaced with Neurobasal medium (Gibco, cat. no. 21103049) + 2% B27 (Gibco, cat. no. 17504044), and half of the medium was replaced every 3- 4 days. On day 8, 10 μM Aβ1-42 fibrils (GL biochem, Cat. No. 53819) and various concentrations of test antibodies were added. After incubation for 3 days, 30 μl/well of Cell Titer-Glo (Promega, Cat #G7571) was added and the plate was read using a microplate reader (PerkinElmer, Vector3) by the chemiluminescent method.

The experimental results are shown in Fig. 2. HAV-2401, HAV-5101, HAV-3601, HAV-9001 and the positive control antibody Aducanumab can protect primary neurons from Aβ fibril toxicity, and a dose-dependent effect was observed in the concentration range of 10-100 µg/mL.

Assay Example 4 Anti-Abeta Antibodies Stimulate Phagocytosis of Aβ Fibrils by BV2 Cells

BV2 cells in logarithmic growth phase (FuDan IBS Cell Center, FH0366) were taken and digested with trypsin, centrifuged at 800 rpm for 5 min. Cell density in a 24-well plate was adjusted with RPMI 1640 (containing 10% FBS) to 1.2×10 ⁵ cells/well/500 μl, shaken thoroughly until a single layer of cells was formed and placed in an incubator at 37°C, 5% CO ₂ at night. After 16 hours, 100 µl of a gradient diluted anti-Abeta antibody or negative control (anti-thrombin antibody against an irrelevant target was used as a negative control, and anti-thrombin antibody was prepared according to the preparation method for H1601-008 as described in WO 2017133673 A1) thoroughly mixed with 10 μM Abeta fibrils labeled with a fluorescent label (diluted with 100 μl of F-12K basic medium), incubated at 37°C for one hour, added to a 24-well plate with 500 μl of medium removed, and incubated at 37°C, 5% CO ₂ for 1 hour. The supernatant was removed and the cells were gently washed three times with PBS pre-chilled at 4°C. And then, a pre-chilled 0.25% solution of Trypsin-EDTA (ethylenediaminetetraacetic acid) (1 x) was added in an amount of 200 μl/well and placed at 4°C for 20 minutes. Trypsin was neutralized with F-12K medium containing 10% FBS and centrifuged, and then the cells were washed three times with pre-chilled PBS, 100 μl of cells were collected and applied to a flow cytometer to read the fluorescence intensity of FITC (Fluorescein isothiocyanate - Fluorescein isothiocyanate).

The results of the experiment are shown in Fig. 3. Both HAB-2401 and HAB-3601 can stimulate phagocytosis of Aβ fibrils by BV2 cells, as can the positive control antibody Aducanumab. A dose-dependent effect was observed in the concentration range from 0.03 to 0.3 µg/ml.

Analysis Example 5. Effect of Anti-Abeta Antibodies on Aβ Fibril Phagocytosis by Rat Primary Microglial Cells

The cerebral cortex was dissected from a newborn SD rat, crushed and filtered to obtain a cell suspension. Cells were resuspended in DMEM containing 20% FBS (Gibco, cat. no. 10099-141), transferred to a T75 culture flask (Corning, cat. no. 3473), pre-chilled PDL (Sigma, cat. no. P0899) and placed in an incubator (37°C, 5% CO ₂ ). The medium was changed every 3 days. On day 8, the culture flask was placed on a shaker and shaken at 200 rpm for 1 hour. The supernatant was cooled, centrifuged, counted, diluted to 5E4/well (5 x 10 ⁴ /well) with DMEM/F12 (GE Cat No. SH30023.01) containing 10% FBS, and plated in a 24-well plate. After 6 hours, the medium was changed to serum-free DMEM/F12 medium and incubated overnight. On day 9, different antibody concentrations or a negative control (an anti-thrombin antibody against an irrelevant target was used as a negative control and an anti-thrombin antibody was prepared according to the preparation method for H1601-008 as described in WO 2017133673 A1) was mixed with 10 μM FITC- labeled (Thermo, cat. No. A30006) Abeta 1-42 (GL biochem, cat. No. 53819), respectively, were incubated at 37°C for 30 minutes and centrifuged in order to wash away excess antibodies. A mixture of antibody and Abeta was added to the cells and incubated at 37°C for 30 minutes. The cells were washed twice with PBS (Hyclone, cat. no. SH30256.01) and trypsin (Gibco, cat. no. 25200-072) was added thereto, placed in a refrigerator at 4° C. for 10 minutes to split the cells. And then, serum was added to terminate the reaction, the cells were collected after the supernatant was removed by centrifugation, pre-cooled PBS was added, and the supernatant was removed by centrifugation, and the cells were washed twice with PBS. The FITC fluorescence intensity was read using a flow cytometer (BD, Verse).

The results of the experiment are shown in Fig. 4. In an experiment on phagocytosis by primary rat microglial cells, HAB-2401 and HAB-3601 can stimulate phagocytosis of Aβ fibrils by primary microglial cells, as can the antibody-positive control Aducanumab. A dose-dependent effect was observed in the concentration range from 0.03 to 0.3 µg/ml.

Analysis Example 6 In Vivo Pharmacological Assay of Anti-Aβ Antibodies The 5* FAD (Five Mutations in Familial Alzheimer Disease) mouse model of Alzheimer's disease was used to evaluate the in vivo efficacy of anti-Aβ antibodies. Five hereditary mutations in the APP and PS1 genes, including three mutations in the APP gene (namely, the Swedish mutation (K670N, M671L), the Florida mutation (I716V) and the London mutation (V717I)) and two mutations in the PS1 gene (namely, M146L and L286V) were overexpressed in 5×FAD mice. Thus, compared to wild-type (WT) mice, 5*FAD mice showed clear cognitive and memory impairments and had significant deposition of Aβ plaques in brain tissue. This assay example was designed to evaluate the effectiveness of test antibodies in improving mouse cognition and reducing Aβ plaque deposition by assaying improved mouse behavior, including nesting ability, and spatial memory (spatial memory in the Morris water maze), as well as by analyzing changes in Aβ plaques in brain tissue.

The 5×FAD mice were provided by Shanghai ChemPartner Co., Ltd. Mice at the age of three months were selected and kept at room temperature 23 plus/minus 2°C, with a 12/12 hour light/dark cycle, and with unlimited food and water intake. All analyzed antibodies were in the human-mouse chimeric form, namely, the constant region(s) of the heavy and light chains were replaced by the mouse constant region(s) in order to avoid ADA (antibody to drug ) caused by prolonged administration of antibodies. The experiment included six groups (n equal to 15 in each group), 4 groups of antibodies to be analyzed, one group of Aducanumab as a positive control and one group of PBS as a negative control. HAB-2401 and HAB-3601 were in the mIgG1 chimeric form, while HAB-5101, HAB-9001 and Aducanumab were in the mIgG2a chimeric form, and they were called HAB-2401-Ch, HAB-3601-Ch, HAB-5101-Ch , HAB-9001-Ch, and Aducanumab-Ch, respectively. HAB-2401-Ch and HAB-3601-Ch were prepared by fusing the heavy chain variable region and the light chain variable region of HAB-2401 and HAB-3601 to the mIgG1 heavy chain constant region (the amino acid of the mIgG1 heavy chain constant region is shown in SEQ ID NO 62) and the mIgG1 light chain constant region (the amino acid sequence of the mIgG1 light chain constant region is shown in SEQ ID NO 63), respectively; HAB-5101-Ch, HAB-9001-Ch and Aducanumab-Ch were prepared by fusing the heavy chain variable region and the light chain variable region of HAB-5101, HAB-9001 and Aducanumab to the mIgG2a heavy chain constant region (mIgG2a heavy chain constant region amino acid sequence shown in SEQ ID NO 64) and the mIgG2a light chain constant region (the amino acid sequence of the mIgG2a light chain constant region is shown in SEQ ID NO 65), respectively. The method for producing antibodies is described in Example 4 of this disclosure. The antibody was injected i.b. in the amount of 15 mg/kg (milligram per kilogram of body weight) once a week, and the introduction was carried out at 13:00 - 16:00, for a total of 12 weeks.

All 5×FAD mice were given an additional injection of the appropriate antibody after the behavioral test (Assay Example 8). The mice were divided into three batches, 5 mice in each group. Samples were taken 24 hours, 72 hours and 7 days after the additional dose to quantify the content of antibodies penetrating the BBB (blood-brain barrier blood-brain barrier) at different time points after antibody treatment. Simultaneously with respect to detection of Aβ deposition (Assay Example 9) and cytokines (Assay Example 10), for groups of the same antibody, all samples taken at different time points after the additional dose were pooled together for data processing.

Analysis Example 7 Mouse Nesting Ability Analysis

Nest building is a mouse instinct and is commonly used as an experimental way to measure the social behavior of a mouse. It has been reported that transgenic mice overexpressing APP such as Tg2576, APPswe/PS1, 3×Tg-AD, 5×FAD (Transl Psychiatry. 2015 May 5;5:e562) exhibited impaired nesting ability to varying degrees. This assay example evaluates the effect of test antibodies on improving the social behavior of 5* FAD transgenic mice by scoring the nesting ability of mice with multiple parameters.

Specific procedures for mouse nesting are described below. At approximately 4:00 pm on the first day of the experiment, a piece of compacted cotton wool was placed in each cage, and each mouse was kept in a separate cage. The densified cotton was weighed before placement in the cage (weight 1) and the mating environment remained unchanged. At 10 o'clock the next morning, the mice were taken out and returned to their original cages. The shape of the nest and the position of the pieces of cotton wool remained unchanged. In the past, a digital camera was used to take photographs of nests made by mice (front view and side view to reflect the true shape and height of the nest). The cotton wool that was not torn to pieces was collected and weighed (weight 2). The percentage of remaining cotton wool was calculated. Based on the photographs, according to the shape, height of the nest, and the degree to which the cotton was broken, for each animal, an average score was obtained from three independent scores provided in parallel, in accordance with the scoring criteria, the scores can be adjusted accordingly depending on the specific situations. The scoring criteria are shown in Table 5:

Photographs of each animal were analyzed by using the Lasso tool in Photoshop CS4 software to calculate relative nest area (percentage of nest area to total area of scattered cotton pieces). The cumulative score of the three parameters (cubic score) was calculated according to the formula below:

Cumulative score of three parameters = Percentage of wool remaining * Percentage of nest area × average score

The results of the analysis are shown in Fig. 6 and FIG. 7. There was some difference between vehicle (solvent blank control) and WT (wild type) which reflects the experimental window of this assay. Whereas the Aducanumab-Ch, HAB-2401-Ch and HAB-9001-Ch groups showed some trend towards improvement compared to the vehicle group.

Analysis Example 8: Water Maze Behavioral Test (Morris Water Maze)

The water maze experiment is a behavioral test commonly used in the field of neuroscience to assess spatial learning and memory ability in rodents. In such an experiment, spatial cues around the pool were used to orient the rodents to find and remember the rescue platform hidden underwater in the pool through training.

The Morris water maze consisted of a white, non-toxic and odorless plastic round pool with a diameter of 120 cm and a height of 50 cm and a transparent platform made of organic glass. The platform had a height of 31 cm and the top surface of the platform had a diameter of 8.5 cm. The pool was filled with water until the water surface level was 1 cm above the platform. The pool was equally divided into four sectors, NE, SE, SW, and NW, and N, NE, E, SE, S, SW, W, and NW were marked at eight points, equally dividing the outer circumference of the circular pool. The platform was placed in the center of sector SW, with a distance of 30 cm from the center of the circle and a distance of 30 cm from the wall of the pool (a schematic representation of the water basin is shown in Fig. 8). The installed filming device was connected to a computer and a monitor. The round pool was placed in a well-lit laboratory. Markers NE, SE, SW, and NW were used as clear visual references. Before the experiment, the water was dotted with an appropriate amount of white plastic particles, achieving a uniform coverage of the water surface, and the water temperature was maintained at 23.0 plus/minus 2.0°C. The water was changed every 5 days.

The water maze experiment included the following modules: flag training, hidden platform, and probe test.

8.1. Flag Workout

The flag was placed above the water on an animal orientation platform. Each experimental animal was placed in water (face to the wall of the pool) at point N, and at the same time video monitoring and video recording were started, for 60 seconds. If the animal failed to climb the platform within 60 s, it was manually guided to the platform and allowed to remain for 20 s before being removed from the maze; if the animal found the platform in 60 s, but remained on the platform for less than 20 s, after the end of the 60 s training, it was forced to remain on the platform for 20 s before being removed from the maze; if the animal found the platform in 60 s and remained on the platform for 20 s, it was removed from the maze. After training, each animal was dried with a towel and returned to the cage. After all animals were trained, a new round of the same training was restarted. In this way, each animal was trained 4 times in one day.

8.2. hidden platform

The platform was hidden about 1 cm below the surface of the water. Animals were trained 4 times a day for a total of 12 days. The location where the mice were placed in the water is shown in FIG. 8. During each training session, the animals were placed with their muzzles against the wall of the pool, and at the same time, video monitoring and video recording were started. Each training session lasted for 60 s. If the animal failed to find the platform within 60 s, it was manually guided to the platform and allowed to remain for 15 s before being led out of the maze; if the animal found the platform in 60 s but remained on the platform for less than 15 s, after the end of the 60 s training, it was forced to remain on the platform for 15 s before being led out of the maze; if the animal found the platform in 60 s and remained on the platform for 15 s, it was taken out of the maze. After training, each animal was dried with a towel and returned to the cage. After all animals were trained, a new round of training was restarted from a different location where the animal was placed in the water.

8.3 Probe test

24 hours after the last workout on day 12, the underwater platform was removed for a probe test. The animal was placed in water (face to the wall of the pool) at the NE point, at the same time, video tracking and video recording were started. After 60 s, tracking ended. The animal was removed from the pool, dried with a towel and returned to the cage. Animal trajectory was analyzed using Noldus Ethovision software. 8.4. Results of the water maze experiment

The learning curve of the hidden platform training is shown in FIG. 9. The results showed that a significant difference was observed between the mouse Tg vehicle group 5*FAD (solvent blank control) and the WT group at day 6, and very significant differences were observed at day 10 and day 11 of hidden platform training, and this difference was generally stable, indicating that there is a difference in spatial memory capacity between 5*FAD transgenic mice and wild-type mice in a behavioral test. This distinction is the basis for testing drug trials; from day 1, the Aducanumab-Ch positive control drug group always showed a shorter delay in reaching the platform than the vehicle group, at day 11, a significant difference was observed, indicating that Aducanumab-Ch showed superior efficacy in this experiment ; Similar to the Aducanumab-Ch group, HAB-2401-Ch, HAB-5101-Ch, and HAB-9001-Ch showed a significant difference at days 11 and 12, indicating that, like Aducanumab-Ch, these three drugs could significantly improve spatial memory and cognitive ability of mice.

In the probe test, the inventors evaluated several experimental parameters, including delay in reaching the platform, delay in reaching the platform area, platform or platform area crossing counts, platform dwell time and platform area dwell time, and ACI index.

The platform delay results are shown in FIG. 10A. There was a definite difference between the WT and the carrier group, which is the experimental window. (In the case of the WT group, the delay in reaching the platform in the probe test was longer than the delay in the flag training, which could be due to experimental variation since only one test was performed in the probe test, while in the flag training it was four tests were carried out). The results for HAB-2401-Ch, HAB-5101-Ch and HAB-9001-ch were comparable to those for the Aducanumab-Ch group, and showed a shorter duration than the vehicle group.

Results for delay in reaching the platform area are shown in FIG. 10V. The WT group showed about three times more significant difference when compared to the vehicle group. The Aducanumab-Ch group showed a significant difference when compared to the vehicle group, while the result of the Aducanumab-Ch group was close to that of the WT group, indicating that positive control molecules can significantly improve the memory of 5*FAD mice. All of the four antibodies analyzed showed significant differences when compared to the vehicle group, among which HAB-9001-Ch showed even better efficacy than Aducanumab-Ch.

The results of the platform or platform zone crossing experiment are shown in FIG. 10C and FIG. 10D, the results of the platform dwell time and platform dwell time experiment are shown in FIG. 10E and FIG. 10F and the results of the ACI index experiment are shown in FIG. 10g. In the probe test, the platform was removed and mouse platform memory is theoretically positively correlated with these parameters. Regarding these five parameters, there was a certain difference between the WT group and the carrier group, indicating the experimental window of the experimental results. Compared to the vehicle group, Aducanumab-Ch showed an improvement trend. Compared to the vehicle group, HAB-2401-Ch, HAB-5101-Ch and HAB-9001-Ch all showed different degrees of improvement, and HAB-9001-Ch showed the best efficacy.

In summary, in the probe test compared to the vehicle group, HAB-2401-Ch, HAB-5101-Ch and HAB-9001-Ch showed different degrees of improvement in delay to reach the platform, delay to reach the platform zone, duration of stay on the platform , platform zone residence time, platform crossing count, platform zone crossing count, and ACI index, indicating that these three antibodies analyzed can significantly improve spatial memory and cognition in the 5* FAD mouse model of Alzheimer's disease.

Analysis Example 9 Detection of Aβ Deposition

All samples were taken from mice used for the behavioral test (Assay Example 8). Each mouse was sprayed with PBS (pH 7.4) and the brain was harvested. The left hemisphere was immediately immersed in 4% PFA (paraformaldehyde) and fixed for 72 hours, and the right hemisphere was dissected on ice to collect the frontal cortex and hippocampus, weighed separately, then flash frozen in liquid nitrogen and stored in a refrigerator at -80 °С for subsequent processing of samples.

The specific procedures for homogenizing the right hemisphere were as follows: the tissue to be analyzed was weighed and transferred to a homogenization tube and 10 times the volume of diethylamine lysate (50 mM NaCl, 0.2% DEA) containing inhibitors was added to it. proteases). Namely, 10 mg of tissue was added to 100 μl of lyse solution, homogenized with a homogenizer, and sonicated in an ice bath for 15-20 seconds. The homogenate was transferred into a centrifuge tube, centrifuged at 100,000 * g at 4°C for 30 min. The resulting supernatant containing soluble Aβ was collected and stored at -80°C. A 10-fold volume of lysing solution of guanidine hydrochloride (5 M GuHCl in PBS) was added to a centrifuge tube, the pellet was resuspended and sonicated in ice water for 15-20 seconds . The sample was centrifuged at 100,000 xg at 4°C for 30 min. The resulting supernatant containing insoluble Aβ was collected and stored at -80°C.

Aβ deposition in the brain tissue homogenate was detected by ELISA (human beta-amyloid ELISA kit (a.k. 1-40) Quantikine: R&D systems, DAB140B; human beta-amyloid ELISA kit (a.k. 1-42 ) Quantikine: R&D systems, DAB142). First, the insoluble components in the brain homogenate (namely, the sample to be analyzed) and the standard were diluted with a diluent; the pre-chilled plate was washed with wash buffer; diluted standard and samples were added; the tablet was sealed and incubated at 4°C for 2 hours; the plate was washed with wash buffer 3-4 times; pre-chilled secondary antibody was added; the tablet was sealed and incubated at 4°C for 2 hours; the plate was washed 3-4 times with wash buffer; the chromogenic substrate solution was added and incubated at room temperature for 30 minutes; stop solution was added and the OD450 value was read on a microplate reader.

The results of the ELISA analysis are shown in FIG. 11A-11D, the vehicle group (solvent blank control) showed significant deposition of Aβ1-40 and Aβ1-42 compared to the WT group, indicating that Aβ deposition was overexpressed in mouse brain tissue by 5 x FAD. Compared to the vehicle group, the Aducanumab-Ch group tended to have lower levels of Aβ1-40 and Aβ1-42. Among the four antibodies analyzed, HAB-3601-Ch had the best potency and tended to have lower levels of insoluble Aβ1-40 (insoluble hippocampal Aβ1-40) and insoluble Aβ1-42 (insoluble hippocampal Aβ1-42) compared to vehicle.

The procedures for immunohistochemical staining of the tissue of the left hemisphere were as follows. Dehydration of the isolated brain: the brain was fixed with 4% PFA for 72 hours, then placed in a 30% sucrose solution for dehydration, placed in a refrigerator at 4°C overnight, and the dehydration procedure was repeated once; tissues were flooded: isopentane was placed in dry ice for several minutes to obtain a liquid in isopentane until reaching -70°C; the brain tissue was dried with blotting paper and placed in isopentane to freeze for a few seconds after removing the excess portion, and then the brain tissue was taken out of isopentane, dried and poured into an OTS (pouring agent) embedding chamber, and then the embedding chamber was placed on a dry ice to obtain a cured OTS. The drenched brain tissue was stored in a refrigerator at -80°C; Section preparation: brain tissue was cut into sagittal sections in a cryostat, 20 μm per section; inactivation of endogenous peroxidase: the brain section was washed 3 times in PBS, 5 minutes each, incubated with 0.3% H ₂ O ₂ (obtained using PBS) for 10 minutes; permeabilization: brain section washed 3 times in PBS, 5 minutes each, incubated with TBST (TBS containing 0.25% Triton X-100) 3 times, 5 minutes each; blocking: incubated with 0.3% Triton X-100 and 5% BSA in PBS for 1 hour; incubation with primary antibody: primary antibody 3D6 was diluted with 2% BSA in TBST to a working concentration of 10 μg/ml, and the brain section was placed in the working solution of the primary antibody and incubated overnight at 4°C; secondary antibody incubation: the brain section was washed in TBST three times for 5 minutes each, the secondary antibody was diluted with TBST containing 2% BSA to obtain a working concentration of 5 μg/ml, the brain section was incubated in the working solution of the secondary antibody for 1 h; color developed: the brain section was washed 3 times in TBST for 5 minutes each, and placed in a solution to develop color in the dark for 5 minutes, and then immersed twice in pure water to stop the color development reaction; medium embedding: brain section was placed in and out of PBS, dried, immersed in pure water to remove salt, dried and fixed with an ethanol concentration gradient (75%, 95%, absolute ethanol), resin-embedded and dried; scanning and quantification: The section was scanned with a digital section scanner (Hamamatsu Nanozoomer S210) to obtain scanned images of the section. For each brain slice photograph, cortical and hippocampal regions are first shown according to mouse brain coordinate and then Aβ-positive signal areas are shown by the BIOTOPIX™ analysis system software. Finally, the proportion of positive signals was calculated for the cerebral cortex and hippocampus, respectively. Namely, the proportion of Aβ-positive signals in the cerebral cortex = the area of Aβ-positive signals in the cerebral cortex/total area of the cerebral cortex; proportion of Aβ-positive signals in the hippocampus=area of Ap-positive signals in the hippocampus/total hippocampal area.

The results of the IHC detection experiments are shown in FIG. 12 and FIG. 13. Compared to the WT group, the vehicle group showed clear deposition of Aβ1-40 and Aβ1-42, consistent with ELISA analysis. Compared to the vehicle group, HAB-3601-Ch, HAB-9001-Ch and Aducanumab-Ch can significantly reduce Aβ deposition, indicating that HAB-3601-Ch and HAB-9001-Ch can significantly reduce Aβ deposition. like Aducanumab-Ch.

Assay Example 10 Effect of Antibodies on Cytokine Release in Brain Tissue

The samples used in this analysis example were the soluble component from the brain homogenate in Analysis Example 9, and the detection method to be used was a high sensitivity electrochemiluminescence analyzer MSD (Meso Scale Discovery). Specific experimental procedures are described in the product manual (U-PLEX Biomarker Group Multiplex Assay Kit (mouse): MSD, N05235A-1). Briefly, a biotinylated capture antibody for each cytokine was bound to each U-PLEX linker, reacted at room temperature for 30 minutes, and then the reaction was terminated; the resulting solutions of U-PLEX-linked antibodies were mixed and applied to the plate U-PLEX, incubated at room temperature with shaking for one hour or 4°C during the night; standard and analyzed samples (namely, soluble components in the brain tissue homogenate) were diluted in accordance with the manual; the coated plate was washed 3 times with PBST or 1×MSD wash buffer (wash reagent 1×MSD), 25 μl of sample diluent and 25 μl of the diluted standard and samples to be analyzed were added, and the plate was sealed and incubated at room temperature with shaking for 1 hours. The coated plate was washed 3 times with PBST or 1×MSD wash buffer, 50 μl of the diluted detection antibody mixture was added, and the plate was tightly capped and incubated with shaking at room temperature for 1 hour. The coated plate was washed 3 times with PBST or 1×MSD wash buffer, and then 2* readout buffer was added. Finally, the plate was read from the MSD instrument.

The experimental results are shown in Fig. 14A-Fig. 14D and FIG. 15A-Fig. 15F. The levels of all cytokines such as TNFα, IFN-γ, IL1β, IL2, IL6, IL12p70, IL4, IL10, MCP-1 and KS were very low and almost reached the lower detection limit of the detection method. There were clear differences between individual mice, thus there was no significant difference between groups. In summary, the four groups of antibodies tested did not show higher levels of cytokines compared to the positive control, Aducanumab, indicating that the antibodies tested were safe.

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SEQUENCE LIST

<110> JIANGSU HENGRUI MEDICINE CO., LTD.; SHANGHAI HENGRUI PHARMACEUTICAL CO.,

Ltd.

<120> ANTIBODY AGAINST BETA-AMYLOID, ITS ANTIGEN-BINDING FRAGMENT AND THEM

APPLICATION

<130> 719048CPCT

<140> PCT/CN2019/096159

<141> 07/16/2019

<150> CN201810782196.2

<151> 07/17/2018

<160> 73

<170> SIPOSequenceListing 1.0

<210> 1

<211> 40

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> 1-40 amino acid sequence of Abeta

<400> 1

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys

1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile

20 25 30

Gly Leu Met Val Gly Gly Val Val

35 40

<210> 2

<211> 42

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> 1-42 amino acid sequence of Abeta

<400> 2

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys

1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile

20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala

35 40

<210> 3

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> heavy chain variable region amino acid sequence

mAb-2401

<400> 3

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

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asn Thr

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Thr Asn Gly Asn Thr Lys Tyr Ala Pro Lys Phe

50 55 60

Lys Asp Lys Ala Thr Ile Ile Ala Asp Thr Ser Ser Asn Thr Gly Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

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

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 4

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> light chain variable region amino acid sequence

mAb-2401

<400> 4

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

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

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

50 55 60

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

65 70 75 80

Ile Ser Asn Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Asn Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 5

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> heavy chain variable region amino acid sequence

mAb-3601

<400> 5

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala

50 55 60

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

65 70 75 80

Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Arg Pro Leu Gly Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 6

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> light chain variable region amino acid sequence

mAb-3601

<400> 6

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

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

100 105 110

<210> 7

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> heavy chain variable region amino acid sequence

mAb-5101

<400> 7

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Thr Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Tyr Trp Asp Asp Val Ser Leu Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Glyn Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 8

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> light chain variable region amino acid sequence

mAb-5101

<400> 8

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Cys Gln Gly

85 90 95

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

100 105 110

<210> 9

<211> 124

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> heavy chain variable region amino acid sequence

mAb-9001

<400> 9

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala

50 55 60

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

65 70 75 80

Phe Leu Lys Ile Ala Ile Val Asp Thr Ala Asp Ile Ala Thr Tyr Tyr

85 90 95

Cys Val Arg Arg Gly Phe His Leu Gly Ser Arg Gly Asp Tyr Phe Asp

100 105 110

His Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 10

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> light chain variable region amino acid sequence

mAb-9001

<400> 10

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

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

100 105 110

<210> 11

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> complementarity determining amino acid sequence

heavy chain regions (HCDR1) HAB-2401, mAb-2401

<400> 11

Asn Thr Tyr Met His

fifteen

<210> 12

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence HCDR2 HAB-2401, mAb-2401

<400> 12

Arg Ile Asp Pro Thr Asn Gly Asn Thr Lys Tyr Ala Pro Lys Phe Lys

1 5 10 15

asp

<210> 13

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HCDR3 HAB-2401, mAb-2401

<400> 13

Arg Val Tyr Tyr Tyr Asp Ser Thr Tyr Asn Tyr

1 5 10

<210> 14

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> complementarity determining amino acid sequence

light chain regions (LCDR1) HAB-2401, mAb-2401

<400> 14

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Thr

<210> 15

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> LCDR2 amino acid sequence HAB-2401, mAb-2401

<400> 15

Trp Ala Ser Thr Arg Glu Ser

fifteen

<210> 16

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> LCDR3 amino acid sequence HAB-2401, mAb-2401

<400> 16

Gln Asn Asp Tyr Asn Tyr Pro Leu Thr

fifteen

<210> 17

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence of HCDR1 HAB-9001, mAb-3601, mAb-9001,

HAB-3601,

<400> 17

Thr Phe Gly Met Gly Val Gly

fifteen

<210> 18

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence HCDR2 mAb-9001, mAb-3601

<400> 18

His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala Leu Lys Ser

1 5 10 15

<210> 19

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence HCDR3 HAB-3601, mAb-3601

<400> 19

Arg Pro Leu Gly Ser Tyr Asp Tyr Phe Asp Tyr

1 5 10

<210> 20

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> LCDR1 amino acid sequence HAB-9001, mAb-3601, mAb-5101,

mAb-9001, HAB-3601, HAB-5101

<400> 20

Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 21

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> LCDR2 amino acid sequence HAB-9001, mAb-3601, mAb-5101,

mAb-9001, HAB-3601, HAB-5101

<400> 21

Lys Val Ser Asn Arg Phe Ser

fifteen

<210> 22

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> LCDR3 amino acid sequence HAB-9001, mAb-3601, mAb-9001,

HAB-3601

<400> 22

Phe Gln Gly Ser Arg Val Pro Leu Thr

fifteen

<210> 23

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence HCDR1 HAB-5101, mAb-5101

<400> 23

Thr Ser Gly Met Gly Val Ser

fifteen

<210> 24

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence HCDR2 HAB-5101, mAb-5101

<400> 24

His Ile Tyr Trp Asp Asp Val Ser Leu Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 25

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence HCDR3 HAB-5101, mAb-5101

<400> 25

Arg Arg Ile Ile Thr Val Val Asp Ala Met Asp Tyr

1 5 10

<210> 26

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> LCDR3 mAb-5101 amino acid sequence

<400> 26

Cys Gln Gly Ser Arg Val Pro Pro Thr

fifteen

<210> 27

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence HCDR3 HAB-9001, mAb-9001

<400> 27

Arg Gly Phe His Leu Gly Ser Arg Gly Asp Tyr Phe Asp His

1 5 10

<210> 28

<211> 98

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence of the germline heavy chain template

human lines (IGHV1-24*01)

<400> 28

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

1 5 10 15

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

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Thr

<210> 29

<211> 95

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence of the germline light chain template

human lines (IGKV1-27*01)

<400> 29

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 Gly Ile Ser Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro

85 90 95

<210> 30

<211> 450

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-2401 heavy chain amino acid sequence

<400> 30

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Thr Asn Gly Asn Thr Lys Tyr Ala Pro Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg Val Tyr Tyr Tyr Asp Ser Thr Tyr Asn Tyr Trp Gly Lys

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Lys

450

<210> 31

<211> 220

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-2401 light chain amino acid sequence

<400> 31

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 Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

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

50 55 60

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

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

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

115 120 125

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

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 32

<211> 98

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence of the germline heavy chain template

human lines (IGHV3-30*01)

<400> 32

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 Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg

<210> 33

<211> 95

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence of the germline light chain template

line man (IGKV1-39*01)

<400> 33

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 Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

<210> 34

<211> 451

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-3601 heavy chain amino acid sequence

<400> 34

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 Ala Phe Ser Thr Phe

20 25 30

Gly Met Gly Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ala His Ile Trp Trp Asp Asp Asn Lys Tyr Tyr Asn Pro Ala

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Arg Pro Leu Gly Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Pro Gly Lys

450

<210> 35

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-3601 light chain amino acid sequence

<400> 35

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 Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Ser Arg Val Pro Leu Thr Phe Gly Gly 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> 36

<211> 100

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence of the germline heavy chain template

human lines (IGHV2-70D*04)

<400> 36

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys Phe Tyr Ser Thr Ser

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile

100

<210> 37

<211> 101

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence of the germline light chain template

human lines (IGKV2-40*01)

<400> 37

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ile Glu Phe Pro

100

<210> 38

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-5101 heavy chain amino acid sequence

<400> 38

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Tyr Trp Asp Asp Val Ser Leu Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 39

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-5101 light chain amino acid sequence

<400> 39

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Ser Arg Val Pro Pro Thr Phe Gly Gly 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> 40

<211> 454

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-9001 heavy chain amino acid sequence

<400> 40

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asn Lys Tyr Tyr Asn Pro Ala

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Arg Gly Phe His Leu Gly Ser Arg Gly Asp Tyr Phe Asp

100 105 110

His Trp Gly Gln Gly Thr Thr 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 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 Lys

450

<210> 41

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-9001 light chain amino acid sequence

<400> 41

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser Arg Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu 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> 42

<211> 330

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> heavy chain constant region amino acid sequence

HAB-2401, HAB-3601, HAB-5101, HAB-9001

<400> 42

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 Lys

325 330

<210> 43

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> light chain constant region amino acid sequence

HAB-2401, HAB-3601, HAB-5101, HAB-9001

<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> 120

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> heavy chain variable region amino acid sequence

HAB-2401

<400> 44

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Thr Asn Gly Asn Thr Lys Tyr Ala Pro Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg Val Tyr Tyr Tyr Asp Ser Thr Tyr Asn Tyr Trp Gly Lys

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 45

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> light chain variable region amino acid sequence

HAB-2401

<400> 45

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 Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

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

50 55 60

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

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 46

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> heavy chain variable region amino acid sequence

HAB-3601

<400> 46

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 Ala Phe Ser Thr Phe

20 25 30

Gly Met Gly Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ala His Ile Trp Trp Asp Asp Asn Lys Tyr Tyr Asn Pro Ala

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Arg Pro Leu Gly Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly

100 105 110

Lys Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 47

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> light chain variable region amino acid sequence

HAB-3601

<400> 47

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 Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 48

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> heavy chain variable region amino acid sequence

HAB-5101

<400> 48

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Tyr Trp Asp Asp Val Ser Leu Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

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

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 49

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> light chain variable region amino acid sequence

HAB-5101

<400> 49

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Ser Arg Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 50

<211> 124

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> heavy chain variable region amino acid sequence

HAB-9001

<400> 50

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asn Lys Tyr Tyr Asn Pro Ala

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Arg Gly Phe His Leu Gly Ser Arg Gly Asp Tyr Phe Asp

100 105 110

His Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 51

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> light chain variable region amino acid sequence

HAB-9001

<400> 51

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

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

100 105 110

<210> 52

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> amino acid sequence HAB-3601, HAB-9001 HCDR2

<400> 52

His Ile Trp Trp Asp Asp Asn Lys Tyr Tyr Asn Pro Ala Leu Lys Ser

1 5 10 15

<210> 53

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-5101 LCDR3 amino acid sequence

<400> 53

Ser Gln Gly Ser Arg Val Pro Pro Thr

fifteen

<210> 54

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<221> Gene

<223> HAB-2401 heavy chain nucleotide sequence

<400> 54

60

agctgcaagg tgagcggcta caccctgacc aacacctaca tgcactggggt gaggcaggcc 120

cctgggaagg gcctggagtg gatgggcagg atcgacccca ccaacggcaa caccaagtac 180

gcccccaagt tcaaggacag ggtgaccatg accgccgaca ccagcaccga cactgcgtac 240

atggagctga gcagcctgag aagcgaggac accgccgtgt actactgcgc caggagggtc 300

tactactacg acagcaccta taactactgg ggtaagggca ccactgtaac cgtgagctct 360

gcttcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag caccctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tcggggtaaa 1350

<210> 55

<211> 660

<212> DNA

<213> Artificial sequence

<220>

<221> Gene

<223> HAB-2401 light chain nucleotide sequence

<400> 55

gacatccaga tgacccaaag ccccagctcc ctgagcgcca gcgtggggga cagggtgact 60

atcacctgca agagcagcca gagcctgctg aactcaggca accagaagaa ctacctgacc 120

tggtatcagc agaagcccgg caaggtgccc aagcttctga tctactgggc cagcaccagg 180

gagagcggcg tgcccagcag gttcagtggc agcggtagcg gaaccgactt cacccttaca 240

atctcaagcc tgcagcccga ggacgttgcc acctactact gccagaacga ctacaactac 300

cccctcacct tcggcgggagg gactaaggtg gagatcaagc gtacggtggc tgcaccatct 360

gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420

ctgctgaata acttctatcc cagaggcc aaagtacagt ggaaggtgga taacgccctc 480

caatcgggta actccgga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540

ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600

gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660

<210> 56

<211> 1353

<212> DNA

<213> Artificial sequence

<220>

<221> Gene

<223> HAB-3601 heavy chain nucleotide sequence

<400> 56

caggtgcagc tggtggagag cggcggtggc gtggtgcaac ccggcaggag cctgaggctg 60

agctgcgctg ccagcggctt cgccttcagc accttcggca tgggcgtggg ctgggtgagg 120

180

tacaatccag ccctgaagag caggttcacc atcagcaggg acaacagcaa aaacaccctg 240

tacctgcaga tgaataccct gagggccgag gataccgccg tgtactactg cgccaggagg 300

ccctgggct ccctacgatta cttcgactac tggggcaagg gaaccaccgt gaccgtgagc 360

agcgcttcga ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 420

gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480

tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540

tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 600

acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag 660

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 720

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 840

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 900

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 960

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1020

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1080

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1140

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1260

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320

acgcagaaga gcctctccct gtctccggggt aaa 1353

<210> 57

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<221> Gene

<223> HAB-3601 light chain nucleotide sequence

<400> 57

gacatccaga tgacccagag ccccagcagt ctgagcgcca gcgtgggcga cagggtcacc 60

atcacctgta ggagcagcca gagcatcgtg cacagcaacg gcaacaccta cctggagtgg 120

180

tcaggcgttc ccagtaggtt cagcggaagc ggcagcggga ccgacttcac cctgaccatc 240

agctccctgc agcccgagga cttcgccacc tactactgct tccaggggcag cggggtgcct 300

ctgacctttg gtgggggcac caaggtggag attaagcgta cggtggctgc accatctgtc 360

ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420

ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480

tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 540

agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600

gtcacccatc agggcctgag ctcgcccgtc acaaaagct tcaacagggg agagtgt 657

<210> 58

<211> 1356

<212> DNA

<213> Artificial sequence

<220>

<221> Gene

<223> HAB-5101 heavy chain nucleotide sequence

<400> 58

caggtgaccc tgaaagaaag cggccctgct ctggtgaagc ccacccagac cctcaccctg 60

acctgcacct tcagcggctt cagcttgagc accagcggca tgggcgtgag ctggatcagg 120

cagcctcccg gcaaggccct ggagtggctg gcccacatct actgggacga cgtgagcctg 180

tacaacccta gcctgaagag ccgcctgaca atcagcaagg acaccagcaa gaaccaggtg 240

gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cctactactg cgccaggagg 300

aggatcatca ccgtggtgga cgccatggac tattggggcc agggcaccac tgtgaccgtg 360

agcagcgctt cgaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 420

tctggggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480

gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540

tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 600

660

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 720

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 780

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 840

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 900

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 960

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1020

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1080

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1140

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1200

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1260

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1320

tacacgcaga agagcctctc cctgtctccg ggtaaa 1356

<210> 59

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<221> Gene

<223> HAB-5101 light chain nucleotide sequence

<400> 59

gacgtcgtga tgacccagac ccccctgagc ctgcccgtga cccctggcga gcccgccagc 60

atcagctgca ggagcagcca gagcatcgtg cacagcaacg gcaacaccta cctggagtgg 120

tacctgcaga agcccggtca gagccccaag ctgctgatat acaaggtgag caacaggttc 180

agcggcgtgc ccgacaggtt ttccggcagc ggaagcggca ccgacttcac cctgaagatc 240

agccgcgtgg aggccgagga cgtgggcgtg tactactgca gtcagggaag ccgagtgccc 300

cccacctttg gaggcggaac taaggtggag atcaagcgta cggtggctgc accatctgtc 360

ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420

ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480

tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 540

agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600

gtcacccatc agggcctgag ctcgcccgtc acaaaagct tcaacagggg agagtgt 657

<210> 60

<211> 1362

<212> DNA

<213> Artificial sequence

<220>

<221> Gene

<223> HAB-9001 heavy chain nucleotide sequence

<400> 60

caggtgaccc tcaaggaatc tggccctgca ctggtgaagc ccacccagac cctgacgctg 60

acctgcacct ttagcggctt cagcttgagc accttcggca tgggcgtggg ctggatcagg 120

cagcctcccg gaaaggccct ggagtggctg gcccacatct ggtgggacga caacaagtac 180

tacaaccccg ctctgaagag ccgcctgaca atcagcaagg acaccagcaa gaaccaggtg 240

gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cctactactg cgccaggagg 300

ggcttccacc ttggcagcag gggcgactac ttcgaccact ggggccaagg caccactgtg 360

accgtgagca gcgcttcgac caagggccca tcggtcttcc ccctggcacc ctcctccaag 420

agcacctctg ggggcacagc ggccctggggc tgcctggtca aggactactt ccccgaaccg 480

gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 540

ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 600

ggcaccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 660

aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 720

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 780

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 840

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 900

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 960

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1020

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1080

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1140

cccagcgaca tcgccgtgga gtggggagagc aatgggcagc cggagaacaa ctacaagacc 1200

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1260

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1320

aaccactaca cgcagaagag cctctccctg tctccgggta aa 1362

<210> 61

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<221> Gene

<223> HAB-9001 light chain nucleotide sequence

<400> 61

gacgtcgtga tgacccagac ccccctgagc ctgcccgtga ccccaggaga acccgccagc 60

atcagctgca ggagcagcca gagcatcgtg cacagcaacg gcaacaccta cctggagtgg 120

tacctgcaga agcctggcca aagcccccag ctgctgatat acaaggtgag caacaggttc 180

agcggcgtgc ccgataggtt ctctggcagt ggcagcggga ccgacttcac cctgaagatt 240

agcagagtgg aggccgagga cgtgggcgtg tactactgct tccaaggcag ccgcgtgccc 300

ctgaccttcg gccagggcac taagctggag atcaagcgta cggtggctgc accatctgtc 360

ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420

ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480

tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 540

agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600

gtcacccatc agggcctgag ctcgcccgtc acaaaagct tcaacagggg agagtgt 657

<210> 62

<211> 324

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> heavy chain constant region amino acid sequence

mIgG1

<400> 62

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

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

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Glu Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Pro Arg Pro Ser Glu Thr Val

65 70 75 80

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

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

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

245 250 255

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

260 265 270

Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 63

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> light chain constant region amino acid sequence

mIgG1

<400> 63

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

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

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

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 64

<211> 330

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> heavy chain constant region amino acid sequence

mIgG2a

<400> 64

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile

65 70 75 80

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

85 90 95

Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

100 105 110

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

115 120 125

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

130 135 140

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

145 150 155 160

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

165 170 175

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

180 185 190

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

195 200 205

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

210 215 220

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu

225 230 235 240

Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met

245 250 255

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

260 265 270

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

275 280 285

Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

290 295 300

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

305 310 315 320

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

325 330

<210> 65

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> light chain constant region amino acid sequence

mIgG2a

<400> 65

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

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

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

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 66

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-2401 with heavy chain variable region grafted

<400> 66

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Thr Asn Gly Asn Thr Lys Tyr Ala Pro Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Thr Arg Val Tyr Tyr Tyr Asp Ser Thr Tyr Asn Tyr Trp Gly Lys

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 67

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-2401 with grafted light chain variable region

<400> 67

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 Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

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

50 55 60

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

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 68

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-3601 with heavy chain variable region grafted

<400> 68

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 Thr Phe

20 25 30

Gly Met Gly Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Arg Pro Leu Gly Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly

100 105 110

Lys Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 69

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-3601 with grafted light chain variable region

<400> 69

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 Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 70

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-5101 with heavy chain variable region grafted

<400> 70

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Tyr Trp Asp Asp Val Ser Leu Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

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

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 71

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-5101 with grafted light chain variable region

<400> 71

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Ser Arg Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 72

<211> 124

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-9001 with heavy chain variable region grafted

<400> 72

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala

50 55 60

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

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Arg Gly Phe His Leu Gly Ser Arg Gly Asp Tyr Phe Asp

100 105 110

His Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 73

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<221> domain

<223> HAB-9001 with grafted light chain variable region

<400> 73

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

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

100 105 110

<---

Claims (46)

1. An anti-amyloid beta (Abeta) antibody or antigen-binding fragment thereof, which is any anti-Abeta antibody or antigen-binding fragment thereof, selected from the following A-D: A) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises the heavy chain complementarity determining regions (HDCRs) HCDR1 as shown in SEQ ID NO 11, HCDR2 as shown in SEQ ID NO 12 and HCDR3 as shown in SEQ ID NO 13 and light chain complementarity determining regions (LCDRs) LCDR1 as shown in SEQ ID NO 14, LCDR2 as shown in SEQ ID NO 15, and LCDR3 as shown in SEQ ID NO 16; B) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises heavy chain HCDR1 as shown in SEQ ID NO 17, HCDR2 as shown in SEQ ID NO: 18 or 52, and HCDR3 as shown in SEQ ID NO 19, and LCDR1 as shown in SEQ ID NO 20, LCDR2 as shown in SEQ ID NO 21 and LCDR3 as shown in SEQ ID NO 22; C) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises heavy chain HCDR1 as shown in SEQ ID NO 23, HCDR2 as shown in SEQ ID NO 24 and HCDR3 as shown in SEQ ID NO 25 and LCDR1 as shown in SEQ ID NO 25 and LCDR1 as shown in SEQ ID NO 23. shown in SEQ ID NO 20, LCDR2 as shown in SEQ ID NO 21, and LCDR3 as shown in SEQ ID NO: 26 or 53; and D) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises heavy chain HCDR1 as shown in SEQ ID NO 17, HCDR2 as shown in SEQ ID NO: 18 or 52, and HCDR3 as shown in SEQ ID NO 27, and LCDR1 as shown in SEQ ID NO 20, LCDR2 as shown in SEQ ID NO 21 and LCDR3 as shown in SEQ ID NO 22. 2. An anti-Abeta antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a murine, chimeric, or humanized antibody. 3. An anti-Abeta antibody or antigen-binding fragment thereof according to claim 2, wherein the antibody is a murine antibody and the antibody contains a heavy chain variable region as shown in SEQ ID NO: 3, 5, 7, or 9 or having at least 95% noah sequence identity with them; and/or a light chain variable region as shown in SEQ ID NO: 4, 6, 8 or 10 or having at least 95% sequence identity therewith. 4. An anti-Abeta antibody or antigen-binding fragment thereof according to claim 3, which is any anti-Abeta antibody or antigen-binding fragment thereof selected from the following E-H: E) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region as shown in SEQ ID NO 3 and a light chain variable region as shown in SEQ ID NO 4; F) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region as shown in SEQ ID NO 5 and a light chain variable region as shown in SEQ ID NO 6; G) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region as shown in SEQ ID NO 7 and a light chain variable region as shown in SEQ ID NO 8; and H) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region as shown in SEQ ID NO 9 and a light chain variable region as shown in SEQ ID NO 10. 5. The anti-Abeta antibody or antigen-binding fragment thereof according to claim 2, wherein the antibody is a humanized antibody and the framework region (FR) sequence of the antibody contains a human germline FR region or a variant thereof, the variant FR region has up to 10 amino acid backmutations in the framework light chain and/or heavy chain framework region of a human antibody, respectively. 6. An anti-Abeta antibody or antigen-binding fragment thereof according to claim 5, which is any anti-Abeta antibody or antigen-binding fragment thereof selected from the following a) to d): a) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region as shown in SEQ ID NO 44 or having at least 90% sequence identity therewith and a light chain variable region as shown in SEQ ID NO 44 NO 45 or having at least 90% sequence identity with it; b) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region as shown in SEQ ID NO 46 or having at least 90% sequence identity therewith and a light chain variable region as shown in SEQ ID NO 46 NO 47 or having at least 90% sequence identity with it; c) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region as shown in SEQ ID NO 48 or having at least 90% sequence identity therewith and a light chain variable region as shown in SEQ ID NO 48 NO 49 or having at least 90% sequence identity with it; d) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region as shown in SEQ ID NO 50 or having at least 90% sequence identity therewith and a light chain variable region as shown in SEQ ID NO. NO 51 or having at least 90% sequence identity with it. 7. An anti-Abeta antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody is a humanized antibody and contains variable regions as shown in any of the following: a) an antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO 11, SEQ ID NO 12 and SEQ ID NO 13, respectively, and an FR region(s) containing(s) one or more amino acid reverses mutations selected from the group consisting of 1E, 71A and 94R; and an antibody light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO 14, SEQ ID NO 15, and SEQ ID NO 16, respectively; b) an antibody heavy chain variable region containing HCDR1 and HCDR3 as shown in SEQ ID NO 17 and SEQ ID NO 19, respectively, and HCDR2 as shown in SEQ ID NO: 18 or 52, and FR region(s) containing ( f) one or more amino acid backmutations selected from the group consisting of 28A and 82B T; and an antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, respectively; c) an antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO 23, SEQ ID NO 24 and SEQ ID NO 25, respectively; and an antibody light chain variable region comprising LCDR1 and LCDR2 as shown in SEQ ID NO 20 and SEQ ID NO 21, respectively, and LCDR3 as shown in SEQ ID NO: 26 or 53; and FR region(s) containing(s) one or more amino acid backmutations selected from the group consisting of 2V and 45K; or d) an antibody heavy chain variable region comprising HCDR1 and HCDR3 as shown in SEQ ID NO 17 and SEQ ID NO 27, respectively, and HCDR2 as shown in SEQ ID NO 18 or 52; and an antibody light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO 20, SEQ ID NO 21, and SEQ ID NO 22, respectively, and FR region(s) containing(s) amino acid backmutation 2V. 8. Antibody against Abeta or antigennegative fragment according to any one of paragraphs. 1-7, where the antibody contains the constant(s) region(s); preferably, the amino acid sequence of the antibody heavy chain constant region is as shown in SEQ ID NO 42 or has at least 85% sequence identity therewith, and the amino acid sequence of the antibody light chain constant region is as shown in SEQ ID NO 43, or has at least 85% sequence identity with it. 9. An anti-Abeta antibody or antigen-binding fragment thereof according to claim 8, wherein the antibody contains a heavy chain as shown in SEQ ID NO: 30, 34, 38, or 40 or has at least 85% sequence identity with them, and/ or a light chain as shown in SEQ ID NO: 31, 35, 39 or 41 or having at least 85% sequence identity with them. 10. An anti-Abeta antibody or antigen-binding fragment thereof according to claim 9, which is any anti-Abeta antibody or antigen-binding fragment thereof selected from the following Q-Ts: Q) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain as shown in SEQ ID NO 30 and a light chain as shown in SEQ ID NO 31; R) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain as shown in SEQ ID NO 34 and a light chain as shown in SEQ ID NO 35; S) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain as shown in SEQ ID NO 38 and a light chain as shown in SEQ ID NO 39; and T) an anti-Abeta antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain as shown in SEQ ID NO 40 and a light chain as shown in SEQ ID NO 41. 11. Antibody against Abeta or antigennegative fragment according to any one of paragraphs. 1-10, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, scFv, diabody, dsFv, and an antigen-binding fragment of a peptide containing complementarity determining region (CDR). 12. A nucleic acid molecule encoding an anti-Abeta antibody or antigen-binding fragment according to any one of paragraphs. 1-11. 13. The nucleic acid molecule according to claim 12, which is any nucleic acid molecule selected from the following U-Z: U) a nucleic acid molecule that contains a nucleotide sequence as shown in SEQ ID NO 52, or a nucleotide sequence having at least 85% sequence identity with it, and/or contains a nucleotide sequence as shown in SEQ ID NO 53 , or a nucleotide sequence having at least 85% sequence identity with it; V) a nucleic acid molecule that contains a nucleotide sequence as shown in SEQ ID NO 54, or a nucleotide sequence having at least 85% sequence identity with it, and/or contains a nucleotide sequence as shown in SEQ ID NO 55 , or a nucleotide sequence having at least 85% sequence identity with it; W) a nucleic acid molecule that contains a nucleotide sequence as shown in SEQ ID NO 56, or a nucleotide sequence having at least 85% sequence identity with it, and/or contains a nucleotide sequence as shown in SEQ ID NO 57 , or a nucleotide sequence having at least 85% sequence identity with it; and Z) a nucleic acid molecule that contains a nucleotide sequence as shown in SEQ ID NO 58, or a nucleotide sequence having at least 85% sequence identity with it, and/or contains a nucleotide sequence as shown in SEQ ID NO 59 , or a nucleotide sequence having at least 85% sequence identity with it. 14. A recombinant expression vector for expressing an anti-Abeta antibody or an antigen-binding fragment thereof, containing a nucleic acid molecule according to claim 12 or 13. 15. A host cell for expressing an anti-Abeta antibody or antigen-binding fragment thereof, wherein the host cell comprises the recombinant vector of claim 14, and wherein the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a cell mammal. 16. The method of obtaining antibodies against Abeta or antigennegative fragment according to any one of paragraphs. 1-11, including the steps: culturing the host cell according to claim 15 in a culture medium and then purifying and isolating this antibody. 17. Pharmaceutical composition for the treatment or prevention of a disease or disorder mediated by Abeta, containing: an effective amount of an anti-Abeta antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-11, and one or more pharmaceutically acceptable carriers, excipients or diluents. 18. The use of antibodies against Abeta or antigennegative fragment according to any one of paragraphs. 1-11 or the pharmaceutical composition of claim 17 in the preparation of a medicament for the treatment or prevention of an Abeta-mediated disease or disorder. 19. Use according to claim 18, wherein the disease is a neurodegenerative disease selected from the group consisting of Alzheimer's disease, mild cognitive impairment, frontotemporal dementia, Lewy body disease, Parkinson's disease, Pick's disease, Bevac's disease, congophilic amyloid angiopathy, cerebral amyloid angiopathy, Down's syndrome, multi-infarct dementia, Huntington's disease, Creutzfeldt-Jakob disease, AIDS dementia syndrome, depression, anxiety neurosis, phobia, Bell's palsy, epilepsy, encephalitis, multiple sclerosis, neuromuscular disorder, neurooncological disease, brain tumor brain, neurovascular disorder due to stroke, neuroimmunological disease, neuropathic otological disease, traumatic injury to the nervous system due to spinal cord injury, pain due to neuropathic pain, childhood neuro- and neuropsychiatric disorder, sleep disorders a, Tourette's syndrome, vascular dementia, cystic fibrosis, Gaucher's disease and other dyskinesias, and diseases of the central nervous system.

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