# Patent

## Title
antibodies binding to a C-terminal fragment of apoliopoprotein E

## Abstract

A human antibody fragment, which antibody or fragment: (i) binds to a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 of the C-terminal domain of Apolipoprotein E (ApoE-CTD) or the amino acid sequence of a part thereof; and (ii) binds to human plaques.

## Background
This application is a U.S. national stage of International Patent Application No. PCT/EP2004/013426, filed 26 Nov. 2004, which designated the U.S. and claims priority of U.S. Provisional Application No. 60/525,174, filed 28 Nov. 2003; the entire contents of each of which are hereby incorporated by reference.


FIELD OF THE INVENTION
The present invention relates to antibodies that specifically bind to a C-terminal fragment of Apolipoprotein E (ApoE). The present invention also provides methods for obtaining such polypeptides and the use of such polypeptides in the diagnosis and treatment of Alzheimer's disease, systemic amyloidosis and other amyloid disorders.
BACKGROUND TO THE INVENTION
Amyloidosis is a progressive, incurable metabolic disease of unknown cause characterized by abnormal deposits of protein in one or more organs or body systems. Amyloid proteins are manufactured, for example, by malfunctioning bone marrow. Amyloidosis, which occurs when accumulated amyloid deposits impair normal body function, can cause organ failure or death. It is a rare disease, occurring in about eight of every 1,000,000 people. It affects males and females equally and usually develops after the age of 40. At least 15 types of amyloidosis have been identified. Each one is associated with deposits of a different kind of protein.
The major forms of amyloidosis are primary systemic, secondary, and familial or hereditary amyloidosis. There is also another form of amyloidosis associated with Alzheimer's disease. Primary systemic amyloidosis usually develops between the ages of 50 and 60. With about 2,000 new cases diagnosed annually, primary systemic amyloidosis is the most common form of this disease in the United States. Also known as light-chain-related amyloidosis, it may also occur in association with multiple myeloma (bone marrow cancer). Secondary amyloidosis is a result of chronic infection or inflammatory disease. It is often associated with Familial Mediterranean fever (a bacterial infection characterized by chills, weakness, headache, and recurring fever), Granulomatous ileitis (inflammation of the small intestine), Hodgkin's disease, Leprosy, Osteomyelitis and Rheumatoid arthritis.
Familial or hereditary amyloidosis is the only inherited form of the disease. It occurs in members of most ethnic groups, and each family has a distinctive pattern of symptoms and organ involvement. Hereditary amyloidosis is though to be autosomal dominant, which means that only one copy of the defective gene is necessary to cause the disease. A child of a parent with familial amyloidosis has a 50-50 chance of developing the disease.
Amyloidosis can involve any organ or system in the body. The heart, kidneys, gastrointestinal system, and nervous system are affected most often. Other common sites of amyloid accumulation include the brain, joints, liver, spleen, pancreas, respiratory system, and skin.
Alzheimer's disease (AD) is the most common form of dementia, a neurologic disease characterized by loss of mental ability severe enough to interfere with normal activities of daily living, lasting at least six months, and not present from birth. AD usually occurs in old age, and is marked by a decline in cognitive functions such as remembering, reasoning, and planning.
Between two and four million Americans have AD; that number is expected to grow to as many as 14 million by the middle of the 21st century as the population as a whole ages. While a small number of people in their 40s and 50s develop the disease, AD predominantly affects the elderly. AD affects about 3% of all people between ages 65 and 74, about 20% of those between 75 and 84, and about 50% of those over 85. Slightly more women than men are affected with AD, even when considering women tend to live longer, and so there is a higher proportion of women in the most affected age groups.
Several genes have been implicated in AD, including the gene for amyloid precursor protein, or APP, responsible for producing amyloid. Mutations in this gene are linked to some cases of the relatively uncommon early-onset forms of AD. Other cases of early-onset AD are caused by mutations in the presenilin genes, PS-1 and PS-2. A dementia similar to AD eventually affects nearly everyone with Downs syndrome, caused by an extra copy of chromosome 21. Other mutations on other chromosomes have been linked to other early-onset cases.
Potentially the most important genetic link was discovered in the early 1990s on chromosome 19. A gene on this chromosome, apoE, codes for a protein involved in transporting lipids into neurons.
Apolipoprotein E (ApoE) is a 34 kDa glycosylated protein. The main sites of ApoE production are the liver and brain. ApoE is a constituent of very low density lipoprotein (VLDL), a subclass of high density lipoproteins and chylomicrons. Cellular uptake of lipid complexes is mediated by binding of ApoE to the low density lipoprotein (LDL) receptor and other related receptors.
There are three major ApoE isoforms in humans, apoE2, apoE3 and apoE4 which are products of three alleles, ε2, ε3 and ε4. In the general population, the ε3 allele is the most common, accounting for 78% of all apoE alleles. The frequency of the ε4 allele is increased significantly in the population of late-onset sporadic and familial Alzheimer's disease (AD) patients.
ApoE contains a C-terminal domain (ApoE-CTD) and an N-terminal domain (ApoE-NTD) joined by a random-coil region. The C-terminal domain comprises a lipid binding site and the N-terminal domain binds to lipoprotein receptors. The CTD amino acid sequence is identical in all three isoforms of ApoE. The CTD and NTD may be separated by cleavage with thrombin.
Direct interactions between ApoE and Amyloid β(Aβ) have been demonstrated in vitro. ApoE is also present in AD plaques. It has been reported that the N-terminal domain of ApoE (ApoE-NTD) mediates binding of apoE to Aβ(Golabek et al., (2000) Biophysical Journal 79: 1008-1015). However, AD plaques containing ApoE have been shown to comprise full-length ApoE at the centre of the plaques and a C-terminal domain fragment of ApoE (ApoE-CTD) at the periphery of the plaques (Cho et al., (2001) J. Neuropathology and Expt. Neurology 60: 342-349). Aβ1-42 deposition in plaques has been shown to precede ApoE deposition whilst Aβ1-40 deposition follows ApoE depositon in plaque maturation (Terai et al., (2001), Brain Research 900: 48-56).
The function of ApoE in the brain is not thought to be specific for AD. ApoE appears to play an important role in modifying recovery from acute brain injury. In particular, there is evidence from both clinical and animal studies to suggest that the presence of the ApoE4 isoform is associated with poor neurological recovery from a variety of acute brain injuries.
SUMMARY OF THE INVENTION
The present inventors developed therapeutic antibodies directed to a region of Apolipoprotein E (ApoE) which is exposed in protein aggregates found in amyloid deposits including Alzheimer plaques but which is not accessible, or has only restricted accessibility, in other forms of ApoE such as ApoE in lipoprotein particles in the blood.
Accordingly, the present invention provides:

    
    
        a human antibody or antibody fragment, which antibody or fragment:
    
    


(i) binds to a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 of the C-terminal domain of Apolipoprotein E (ApoE-CTD) or the amino acid sequence of a part thereof; and
(ii) binds to human plaques;

    
    
        a human antibody or antibody fragment, which antibody or fragment:
    
    


(i) binds to a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 of ApoE-CTD or the amino acid sequence of a part thereof; and
(ii) comprises a heavy chain CDR3 region comprising the sequence shown in SEQ ID NO: 20, SEQ ID NO: 512, SEQ ID NO: 513, SEQ ID NO: 514, SEQ ID NO: 515, SEQ ID NO: 516 or SEQ ID NO: 517;

    
    
        a human antibody or antibody fragment, which antibody or fragment:
    
    


(i) binds to a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 of ApoE-CTD or the amino acid sequence of a part thereof; and
(ii) comprises a heavy chain CDR3 region comprising an amino acid sequence selected from the sequences shown in SEQ ID NO: 29, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 86 and SEQ ID NO: 89;

    
    
        a human antibody or antibody fragment, which antibody or fragment binds, in the presence of VLDL, to a polypeptide having the ApoE-CTD amino acid sequence shown in SEQ ID NO: 1 or the amino sequence of a part thereof;
        a human antibody or antibody fragment, which antibody or fragment:
    
    


(i) binds to human plaques; and
(ii) comprises a heavy chain CDR3 region comprising the sequence shown in SEQ ID NO: 20, SEQ ID NO: 512, SEQ ID NO: 513, SEQ ID NO: 514, SEQ ID NO: 515, SEQ ID NO: 516 or SEQ ID NO: 517;

    
    
        a human antibody or antibody fragment, which antibody or fragment:
    
    


(i) binds to human plaques; and
(ii) comprises a heavy chain CDR3 region comprising an amino acid sequence selected from the sequences shown in SEQ ID NO: 29, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 86 and SEQ ID NO: 89;

    
    
        an antibody or antibody fragment which comprises the heavy chain sequence shown in SEQ ID NO: 136 and the light chain sequence shown in SEQ ID NOS: 521 and 522;
        an antibody or antibody fragment which comprises the heavy chain sequence shown in SEQ ID NO: 142 and the light chain sequence shown in SEQ ID NO: 523;
        an antibody or antibody fragment which comprises the heavy chain sequence shown in SEQ ID NO: 40 and the light chain sequence shown in SEQ ID NO: 517 and/or 518;
        an antibody or antibody fragment which comprises the heavy chain sequence shown in SEQ ID NO: 40 and the light chain sequence shown in SEQ ID NO: 519 and/or 520;
        an antibody or antibody fragment which comprises the heavy chain CDR1 sequence shown in SEQ ID NO: 24, the heavy chain CDR2 sequence shown in SEQ ID NO: 25 and the heavy chain CDR3 sequence shown in any one of SEQ ID NOS: 207, 209 and 210;
        an antibody or antibody fragment which comprises the heavy chain CDR1 sequence shown in SEQ ID NO: 48, the heavy chain CDR2 sequence shown in SEQ ID NO: 49 and the heavy chain CDR3 sequence shown in any one of SEQ ID NOS: 320, 322 and 323;
        an antibody or antibody fragment which comprises the heavy chain CDR1 sequence shown in SEQ ID NO: 66, the heavy chain CDR2 sequence shown in SEQ ID NO: 67 and the heavy chain CDR3 sequence shown in SEQ ID NO: 373;
        an antibody or antibody fragment according to any one of the preceding claims which is a monoclonal antibody,
        an antibody or antibody fragment according to the invention, for use in a method of treatment of the human or animal body by therapy or in a diagnostic method practiced on the human or animal body,
        use of an antibody or antibody fragment according to the invention, in the manufacture of a medicament for the treatment or prevention of an amyloid disorder;
        a pharmaceutical composition comprising an antibody or antibody fragment according to the invention and a pharmaceutically acceptable carrier or diluent;
        a method of treating a subject suffering from an amyloid disorder comprising administering to said subject a therapeutically effective amount of an antibody or antibody fragment according to the invention;
        a method of diagnosing an amyloid disorder in a subject comprising:
    
    


(i) administering to said subject an antibody or antibody fragment according to the invention; and
(ii) determining whether or not said antibody or antibody fragment binds to plaques in said subject, wherein binding of said antibody or antibody fragment to plaques is indicative of an amyloid disorder, thereby determining whether the subject has an amyloid disorder;

    
    
        a polynucleotide encoding an antibody or antibody fragment according to the invention;
        a vector comprising a polynucleotide according to the invention;
        a host cell expressing a polypeptide according the invention;
        a virus encoding a polynucleotide according to the invention;
        a kit for detecting ApoE-CTD, which kit comprises an antibody or antibody fragment according to the invention and means for detecting said an antibody or antibody fragment; and
    
    


a method for detecting the presence of ApoE-CTD in a sample from a subject, which method comprises:
(i) contacting a sample taken from a subject with an antibody or antibody fragment according to the invention under conditions that permit binding of the an antibody or antibody fragment to ApoE-CTD; and
(ii) determining whether or not the an antibody or antibody fragment binds to the sample thereby detecting any ApoE-CTD present in the sample.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the binding of known monoclonal antibodies to biotinylated CTD (bCTD) (A) and VLDL (B) by ELISA.
FIG. 2 shows the binding of phage to bCTD (background=0.05) (A) and VLDL (background=0.1) (B).
FIG. 3 shows an example of bCTD (A) and VLDL (B) ELISA on 203 phage clones.
FIG. 4 shows Biacore affinity analysis on bCTD coated of sFab antibodies M27E11 (A), M28B02 (B) and M26 F05 (C) and Biacore affinity analysis with Fab M27E11 coated on the chip (D).
FIG. 5 is a schematic diagram showing the strategy of transfer of Fab to pBh1.
FIG. 6 shows the binding of antibodies 807A-M0028-B02 (M28B02) (A), 807A-M0026-F05 (M26F05) (B) and 807A-M0027-E11 (M27E11) (C) to human CTD, murine CTD and primate CTD.
FIG. 7 shows the binding of control antibody PH1 (A) and 807A-M0028-B02 (M28B02) (B) to bCTD and bNTD.
FIG. 8 shows the binding of control antibody PH1 (A) and 807A-M0028-B02 (M28B02) (B) to coated VLDL.
FIG. 9 shows the results of antibody binding in competition ELISA between coated bCTD (0.05 μg/mI) and an excess of VLDL or CTD in solution. Binding of M27E11 IgG (A), M28B2 IgG (B), control antibody (C) and PH1 IgG (D) is shown.
FIG. 10 shows the Biacore analysis of 807A-M0026-F05 (M26F05) as soluble Fab (top) and IgG (bottom) on a CTD-control chip.
FIG. 11 shows the Biacore analysis of 807A-M0027-E11 (A) and 807A-M0028-B02 (B) as soluble Fab (top) and IgG (bottom) on a CTD-coated chip.
FIGS. 12 and 13 show the binding of bCTD in solution to Fab 807A-M0027-E1 (M27E11) indirectly coupled to a Biacore chip.
FIG. 14 is a schematic diagram showing the strategy used to transfer V-regions from pBh1 to pRmk2a.
FIG. 15 shows the sequences of the CTD peptides.
FIG. 16 shows the selection campaign of Example 21.
FIG. 17 shows the selection campaign of Example 22.
FIG. 18 shows the selection campaign of Example 23.
FIG. 19 shows a ‘cut and paste’ antibody reformatting strategy.
FIG. 20 shows that 807A-M0028-B02 plaque binding in human AD brain sections is not blocked in presence of VLDL. Binding of 807A-M0028-B02 to human amyloid plaques in vitro was not affected by the presence of VLDL indicating that the antibody has very low affinity for CTD in VLDL relative to CTD in plaques.
FIG. 21 shows the results of an in vitro immunohistochemistry (IHC) screen of sFab antibody clones on human AD brain sections. Several antibodies (Fab clones) from different selections were identified that bind to AD plaques by immunohistochemistry. Note that individual clones may have the same short name i.e. E11 but are from different selections and not identical. (C11=807B-M0004-H03; (selection A) E11=807A-M0027-E11; B2=807A-M0028-B02; (selection B) E11=807B-M0083-E11; D10=807B-M0079D10; A3=807B-M0004-A03; A12=807B-M0013-A12)
FIG. 22 shows representative results (duplicate samples) of the concentration dependent binding of 807A-M0028-B02, 807A-M0028-B02.1 and 807A-M0028-B02.2 to human, primate and murine ApoE-CTD.
FIG. 23 shows representative results (duplicate samples) of the concentration dependent binding of 807A-M0028-B02, 807A-M0028-B02.1, 807A-M0028-B02.2, 807B-M0004-H03, 807B-M0004-H03.1, 807B-M0004-A03 and 807B-M0004-A03.1 to human VLDL.
FIG. 24 shows the detection of 807A-M0028-B02 plaque binding in brain tissue sections from APP/PS1 mouse:
a) In vivo binding of 807A-M0028-B024 days after injection;
b) In vivo binding of 807A-M0028-B027 days after injection.
FIG. 25 shows results of a screen of in vivo plaque binding capacity of 807B M0004H03, 807B-M04-A03, 807B-M0079-D10 and 807B-M0009-F06. Immunohistochemistry expression patterns of anti-CTD hIgG clones B807B-M0004H03, 807B-M0004-A03, 807B-M0079-D10 and 807B-M0009-F06 in APP/PS1 mouse brain sections after in vivo administration is shown.
FIG. 26 shows results of an in vitro screen for plaque binding capacity of affinity maturated Fab-clones in human AD brain sections. Wild-type clone 807B-M0004-A03 (wt A03) was compared to affinity maturated clones 807B-M 0118-B09 (BO9), 807B-M 0117-F05 (F05), 807B-M 0117-G01 (G01) and 807B-M 0118-F03 (F03). Amyloid plaques visualized by anti-CTD binding antibodies (Fab clones) on human AD brain sections.
FIG. 27 shows the strategy used for affinity maturation of 807A-M0028-B02, 807B-M0004-H03, 807B-M004-A03, 807B-M0079-D10 and 807B-M0009-F06.



BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1 is the amino acid sequence of the ApoE-CTD.
SEQ ID NO: 2 is the amino acid sequence of peptide 1 (amino acids 1 to 16 of the ApoE-CTD).
SEQ ID NO: 3 is the amino acid sequence of peptide 2 (amino acids 17 to 32 of the ApoE-CTD).
SEQ ID NO: 4 is the amino acid sequence of peptide 3 (amino acids 33 to 48 of the ApoE-CTD).
SEQ ID NO: 5 is the amino acid sequence of peptide 4 (amino acids 49 to 64 of the ApoE-CTD).
SEQ ID NO: 6 is the amino acid sequence of peptide 5 (amino acids 65 to 80 of the ApoE-CTD).
SEQ ID NO: 7 is the amino acid sequence of peptide 6 (amino acids 9 to 24 of the ApoE-CTD).
SEQ ID NO: 8 is the amino acid sequence of peptide 7 (amino acids 25 to 40 of the ApoE-CTD).
SEQ ID NO: 9 is the amino acid sequence of peptide 8 (amino acids 41 to 56 of the ApoE-CTD).
SEQ ID NO: 10 is the amino acid sequence of peptide 9 (amino acids 57 to 72 of the ApoE-CTD).
SEQ ID NO: 11 is the amino acid sequence of peptide 10 (amino acids 73 to 84 of the ApoE-CTD).
SEQ ID NO: 12 is the amino acid sequence of an epitope in peptide 4 (amino acids 53 to 60 of ApoE-CTD).
SEQ ID NO: 13 is the amino acid sequence of an epitope in peptides 4 and 9 (amino acids 57 to 64 of ApoE-CTD).
SEQ ID NO: 14 is the amino acid sequence of an epitope in peptide 9 (amino acids 61 to 68 of ApoE-CTD).
SEQ ID NO: 15 is the amino acid sequence of an epitope in peptides 1 and 6 (amino acids 9 to 16 of ApoE-CTD).
SEQ ID NO: 16 is the amino acid sequence of an epitope in peptides 4 and 8 (amino acids 49 to 56 of ApoE-CTD).
SEQ ID NO: 17 is the amino acid sequence of an epitope in peptides 3 and 8 (amino acids 41 to 48 of ApoE-CTD).
SEQ ID NO: 18 is the amino acid sequence of peptides 1 and 6 (amino acids 1 to 24 of ApoE-CTD).
SEQ ID NO: 19 is the amino acid sequence of peptides 8 and 9 (amino acids 41 to 72 of ApoE-CTD).
SEQ ID NO: 20 is the amino acid sequence of the consensus CDR3 sequence from antibodies 807A-M0027-E11 and 807A-M0028-B02.
SEQ ID NOS: 21 to 164 are described in Table 8.
SEQ ID NO: 165 is the amino acid sequence of human ApoE4.
SEQ ID NO: 166 is the amino acid sequence of human ApoE3.
SEQ ID NO: 167 is the amino acid sequence of human ApoE2.
SEQ ID NO: 168 is the amino acid sequence of the mature form of human ApoE4.
SEQ ID NO: 169 is the amino acid sequence of the mature form of human ApoE3.
SEQ ID NO: 170 is the amino acid sequence of the mature form of human ApoE2.
SEQ ID NOS: 171 to 206 are described in Table 8.
SEQ ID NOS: 207 to 511 are described in Tables 38 to 42.
SEQ ID NO: 512 is the consensus amino acid sequence of the CDR3 regions of affinity matured clones of 807A-M0028-B02.
SEQ ID NO: 513 is the consensus amino acid sequence of the CDR3 regions of affinity matured clones of 807B-M0004-A03.
SEQ ID NO: 514 is the consensus amino acid sequence of the CDR3 regions of affinity matured clones of 807B-M0004-H03.
SEQ ID NO: 515 is the consensus amino acid sequence of the CDR3 regions of affinity matured clones of 807B-M0009-F06.
SEQ ID NO: 516 is the consensus amino acid sequence of the CDR3 regions of selected affinity matured clones of 807A-M0028-B02.
SEQ ID NO: 517 is the consensus amino acid sequence of the CDR3 regions of selected affinity matured clones of 807B-M0004-A03.
SEQ ID NO: 518 to 527 are defined in Table 21.
DETAILED DESCRIPTION OF THE INVENTION
A. Polypeptides
The present invention provides antibodies that bind to a region on Apolipoprotein E (ApoE) which is exposed in the protein aggregates found in amyloid deposits such as Alzheimer plaques, but which is not present or accessible in other forms of ApoE, such as in lipoprotein particles in the blood.
For the purposes of this invention, the term “antibody”, unless specified to the contrary, includes antibody fragments.
Typically, an antibody of the invention binds the C-terminal domain of Apolipoprotein E (ApoE-CTD), i.e. is reactive with ApoE-CTD. An antibody of the invention does not bind to the N-terminal domain of Apolipoprotein E (ApoE-NTD). The antibody typically binds to the form of ApoE present in human plaques in preference to the form of ApoE present in VLDL. Generally, the form of ApoE present in human plaques is ApoE-CTD. An antibody of the invention preferably binds ApoE-CTD in the presence of very low density lipoprotein (VLDL). An antibody of the invention may be one that binds to an epitope in ApoE-CTD, which epitope is not present in ApoE associated with VLDL. For example, the epitope may be one which is one not accessible or exposed to the antibody when ApoE is associated with VLDL. The epitope to which the antibody binds may typically be hidden in full-length ApoE present in VLDL and so the affinity of the antibody for ApoE is substantially less than its affinity for ApoE-CTD. The epitope to which the antibody binds is present only in ApoE-CTD and not in ApoE-NTD and so the antibody is typically devoid of binding to ApoE-NTD. Any binding of the antibody to ApoE-NTD is generally non-specific binding of a substantially lower affinity than the specific binding of the antibody to ApoE-CTD. A substantially lower affinity is generally at least a two fold, three fold, five fold, 10 fold, 50 fold or 100 fold lower affinity.
An antibody of the invention thus preferentially binds or specifically binds to ApoE-CTD. An antibody “preferentially binds” or “specifically binds” to ApoE-CTD when it binds with preferential or high affinity to ApoE-CTD but does not substantially bind, does not bind or binds with only low affinity to other polypeptides. A variety of protocols for binding, competitive binding or immuno-radiometric assays to determine the specific binding capability of an antibody are well known in the art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226, 1993). Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation. Typically an antibody of the invention, is capable of binding to ApoE-CTD having the sequence shown in SEQ ID NO: 1 with an affinity constant of at least 107 M−1, preferably at least 108 M−1, 109 M−1 or 1010 M−1. An antibody of the invention, is preferably capable of preferentially binding to ApoE-CTD with an affinity that is at least two-fold, 10-fold, 50-fold, 100-fold or greater than its affinity for binding to a non-specific polypeptide such as BSA, casein, VLDL, ApoE-NTD or ApoE present in VLDL.
An antibody which specifically binds to ApoE-CTD typically displays at least 2× background binding in an ELISA on immobilised ApoE-CTD but less than 2× background, typically 1× background, to control proteins such as ApoE-NTD or streptavidin.
An antibody of the invention generally binds to human plaques. The term “human plaques” is intended to cover any amyloid deposits comprising at least one protein having an amino acid sequence encoded by a human gene. Preferably the human plaque is present in or derived from human tissue. More preferably the human plaque is present in a sample that has been obtained from a human subject. The human subject may have an amyloid disorder, such as systemic amyloidosis or Alzheimer's disease. The sample may be taken from any tissue or organ containing amyloid plaques. Suitable tissues and organs include brain, tongue, intestines, skeletal muscle, smooth muscle, nerves, skin, ligaments, heart, liver, spleen and kidneys. Where the subject has Alzheimer's disease, the sample is generally a brain section. The brain section is typically obtained post-mortem. Fibrils prepared from any such sample are also included within the term “human plaques”.
The human plaque may be present in or derived form a non-human animal which is transgenic for one or more, for example two or three, human proteins, which human protein(s) is/are found in amyloid deposits. The human protein is preferably ApoE but may be amyloid precursor protein (APP) (typically comprising the Swedish mutation) or presenilin.
Binding to human plaques may be determined by any suitable method. For example in an IHC assay, binding of an antibody to human plaques can be said to occur when a positive blind scored IHC signal is obtained after staining with <20 μg/ml antibody in two amyloid deposit samples primarily tested or if one sample is negative in the primary test, at least two out of three samples subsequently tested indicates that an antibody binds to human plaques. The samples are preferably derived from different individuals and sectioned from tissue samples with histologically verified amyloid deposits which are IHC positive for an amyloid marker such as Aβ.
The ability of an antibody to bind to human plaques may be determined in vivo using a mouse or other non-human animal model, such as a rodent or primate, of Alzheimer's disease or systemic amyloidosis.
In such an assay, binding of an antibody to plaques may be determined using IHC. The antibody may be labelled prior to being tested. Binding to the plaque may be defined as positive blind scored IHC staining of amyloid after injection of ≦1 mg antibody, in single or multiple doses, in at least two out of three mice tested. The signal is generally compared to the signal from stained anatomically, sex and age matched tissue from negative isotype matched control antibody injected mice.
The term “epitope” as used herein refers to that portion of a molecule that makes contact with a particular binding polypeptide. An epitope may be linear, comprising an essentially linear amino acid sequence from the antigen or conformational, comprising sequences that are separated by other sequences but come together structurally to form a binding site for the polypeptide.
The epitope in ApoE-CTD to which the antibody binds may appear on ApoE-CTD after cleavage from full-length ApoE. Alternatively the epitope may appear following the interaction of ApoE-CTD with amyloid plaques, for example as a result of binding of ApoE-CTD to Aβ. Cleavage of ApoE and/or binding of ApoE-CTD to amyloid plaques may result in the exposure of new linear (peptide) epitopes and/or to the exposure or formation of new conformational epitopes. The epitope to which a polypeptide of the invention binds may be hidden in VLDL-associated ApoE due to the interaction of ApoE with other components of VLDL. The polypeptide may bind specifically to a complex formed between ApoE-CTD and Aβ.
The amino acid sequence of ApoE-CTD is shown in SEQ ID NO: 1. ApoE-CTD epitopes may thus be formed by a linear or conformational sequence within the sequence of ApoE-CTD as shown in SEQ ID NO: 1. An antibody that binds to ApoE-CTD typically bind to an ApoE-CTD polypeptide having the whole sequence shown in SEQ ID NO: 1 but may also bind to a part of the amino acid sequence of SEQ ID NO: 1 such as to a peptide having an amino acid sequence as shown in any one of SEQ ID NOS: 2 to 19. Preferably, the antibody binds to one or more of the peptides shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. The part of ApoE-CTD to which the antibody binds is at least a three amino acid fragment of SEQ ID NO: 1, preferably at least a five, six, seven or eight amino acid fragment, more preferably a 10, 12 or 16 amino acid fragment.
The polypeptide (or peptide) to which the antibody binds may be a recombinant polypeptide. The polypeptide may be in solution or, more preferably, may be attached to a solid surface. For example, the polypeptide may be attached to beads, such as magnetic beads.
The polypeptide may be biotinylated. The biotin molecule conjugated to the peptide may be used to immobilize the polypeptide on a solid surface by coupling biotin to streptavidin on the solid surface.
An antibody of the present invention suitable for use in treating or preventing Alzheimer's disease and/or systemic amyloidosis typically tests positive in an ex vivo phagocyte assay. A positive phagocyte assay is defined as positive blind scored confocal microscopy detection of phagocytes that contain amyloid after co-culture on amyloid tissue after applying ≦20 μg/ml of the antibody in at least two out of three cultures tested. The signal is generally compared to the signal from identical co-cultures containing the same concentration of a negative control antibody. A positive phagocyte assay generally also results in the degradation of amyloid, for example as shown by Western blot to be less than one third the density of the Aβ-based remaining after up to three days of co-culture, as compared to blots from identical co-cultures containing the same concentration of a negative-control antibody.
Antibodies and other peptides for therapeutic use are typically of high affinity, preferably having an affinity of <1 nM, for ApoE-CTD, to enable them to function optimally even at the low concentrations in the brain that will build up after systemic injection.
The term “antibody” refers to a protein comprising at least one, and preferably two, heavy chain variable regions (VH) and/or at least one, preferably two, light chain variable regions (VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions (CDR)”, interspersed with regions that are more conserved, termed “framework regions (FR)”. The extent of the FR and CDRs has been precisely defined (see, Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al. (1987) J. Mol. Biol. 196: 901-917, which are incorporated herein by reference in their entirety). Each VH and VL is composed of three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain respectively. In one embodiment, the antibody is a tetramer of two heavy and two light chains, wherein the heavy and light chains are interconnected by, for example, disulphide bonds. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues and factors, including various cells of the immune system and the first component of the complement system. The term “antibody” includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM and subtypes thereof. A preferred immunoglobulin is IgG.
As used herein the term “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognised human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu conatant region genes as well as a myriad of immunoglobulin variable region genes. Full-length immunoglobulin light chains (about 25 kD or 214 amino acids) are encoded by a variable region gene at the N-terminus (about 110 amino acids) and a kappa or lambda constant region at the C-terminus. Full-length immunoglobulin heavy chains (about 50 kD or 446 amino acids) are encoded by a variable region gene at the N-terminus (about 116 amino acids) and one of the other aforementioned constant region genes at the C-terminus, e.g. gamma (encoding about 330 amino acids).
An antibody fragment of the invention is typically an antigen-binding fragment. The term “antigen-binding fragment” refers to one or more fragments of a full-length antibody that are capable of specifically binding to ApoE-CTD. Examples of binding fragments include (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region; (iii) a Fd fragment (consisting of the VH and CH1 domains); (iv) a Fv fragment (consisting of the VH and VL domains of a single arm of an antibody); (v) a dAb fragment (consisting of the VH domain); (vi) an isolated CDR; (vii) a single chain Fv (scFv) (consisting of the VH and VL domains of a single arm of an antibody joined by a synthetic linker using recombinant means such that the VH and VL domains pair to form a monovalent molecule); (viii) diabodies (consisting of two scFvs in which the VH and VL domains are joined such that they do not pair to form a monovalent molecule; the VH of each one of the scFv pairs with the VL domain of the other scFv to form a bivalent molecule); (ix) bi-specific antibodies (consisting of at least two antigen binding regions, each region binding a different epitope). Preferably, the antibody fragment is a Fab fragment or single-chain antibody (scFv).
The sequences of preferred CDR1 domains are shown in SEQ ID NOS: 21, 24, 27, 30, 33, 36, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 93, 111, 117 and 123. Other preferred CDR1 domains are variants of these sequences in which one or more amino acids within the sequence have been deleted or, more preferably, substituted. Other preferred CDR1 domains are variants of the sequences shown in any one of SEQ ID NOS: 21, 24, 27, 30, 33, 36, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 93, 111, 117 and 123 in which one or more amino acid has been inserted. Preferably, a variant CDR1 domain comprises one or more, for example two, three, four or five substitutions, preferably conservative substitutions. Examples of such CDR1 variant sequences are the LV-CDR1 sequences identified in Tables 38, 39, 40, 41 and 42. Preferred CDR1 sequences include SEQ ID NOS: 33, 219, 226, 218, 326, 93, 325, 391 and 394.
The sequences of preferred CDR2 domains are shown in SEQ ID NOS: 22, 25, 28, 31, 34, 37, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 94, 112, 118 and 124. Other preferred CDR2 domains are variants of these sequences in which one or more amino acids within the sequence have been deleted or, more preferably, substituted. Other preferred CDR2 domains are variants of the sequences shown in one of SEQ ID NOS: 22, 25, 28, 31, 34, 37, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 94, 112, 118 and 124 in which one or more amino acid has been inserted. Preferably, a variant CDR2 domain comprises one or more, for example two, three, four or five substitutions, preferably conservative substitutions. Examples of such CDR2 variant sequences are the LV-CDR2 sequences identified in Tables 38, 39, 40, 41 and 42. Preferred CDR2 sequences include SEQ ID NOS: 382, 386, 333, 334, 34, 247 and 252.
The sequences of preferred CDR3 domains are shown in SEQ ID NOS: 23, 26, 29, 32, 35, 38, 47, 50, 53, 56, 59, 62, 65, 65, 68, 71, 74, 77, 80, 83, 86, 89, 95, 113, 119 and 125. Other preferred CDR3 domains are variants of these sequence in which one or more amino acids within the sequence have been deleted or, more preferably substituted. Other preferred CDR3 domains are variants of the sequences shown in one of SEQ ID NOS: 23, 26, 29, 32, 35, 38, 47, 50, 53, 56, 59, 62, 65, 65, 68, 71, 74, 77, 80, 83, 86, 89, 95, 113, 119 and 125 in which one or more amino acid has been inserted. Preferably, a variant CDR3 domain comprises one or more, for example two or three, conservative substitutions.
Conservative substitutions are shown in the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

















ALIPHATIC
Non-polar
G A P





I L V




Polar-uncharged
C S T M





N Q




Polar-charged
D E





K R



AROMATIC

H F W Y










Examples of variant CDR3 sequences are the HV-CDR3 and LV-CDR3 sequences identified in Tables 38, 39, 40, 41 and 42. Preferred variant CDR3 sequences are shown in SEQ ID NOS: 207, 209, 210, 35, 269, 252, 34, 322, 323, 320, 341, 373 and 378.
Preferred antibodies comprise (i) the VH sequence shown in SEQ ID NO: 39, or a variant thereof, and the VL sequence shown in SEQ ID NO: 42, or a variant thereof; (ii) the VH sequence shown in SEQ ID NO: 40, or a variant thereof, and the VL sequence shown in SEQ ID NO: 43, or a variant thereof; (iii) the VH sequence shown in SEQ ID NO: 41, or a variant thereof, and the VL sequence shown in SEQ ID NO: 44, or a variant thereof.
Other preferred antibodies comprise a heavy chain sequence selected from the sequences shown in SEQ ID NOS: 317, 318, 319, 369, 370, 371, 372 and 397 and optionally a light chain sequence selected from SEQ ID NOS: 43, 295, 294, 304, 347, 348, 357, 362, 406 and 418. More preferred antibodies include antibodies having the following combinations of heavy and light chain sequences: SEQ ID NOS: 319 and 43, SEQ ID NOS: 318 and 295, SEQ ID NOS: 318 and 294, SEQ ID NOS: 317 and 304, SEQ ID NOS: 370 and 347, SEQ ID NOS: 370 and 348, SEQ ID NOS: 371 and 348, SEQ ID NOS: 372 and 348, SEQ ID NOS: 369 and 357, SEQ ID NOS: 370 and 362, SEQ ID NOS: 397 and 406. SEQ ID NOS: 397 and 418.
Variant antibodies may be obtained by any suitable method. Typically variants with improved binding characteristics are selected by affinity maturation.
In a preferred embodiment, the antibody is a recombinant or modified anti-ApoE-CTD antibody, e.g. a chimeric, humanised, deimmunised or an in vitro generated antibody. The term “recombinant” or “modified” antibody as used herein is intended to include all antibodies that are prepared, expressed, created or isolated by recombinant means, such as (i) antibodies expressed using a recombinant expression vector transfected into a host cell; (ii) antibodies isolated from a recombinant, combinatorial antibody library; (iii) antibodies isolated from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes; or (iv) antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies include humanised, CDR grafted, chimeric, deimmunised, in vitro generated antibodies and may optionally include constant regions derived from human germline immunoglobulin sequences.
An antibody according to the invention is a human antibody. The antibody may be a chimeric antibody, a recombinant antibody, a humanised antibody, a monoclonal antibody or a polyclonal antibody. Preferably the antibody is monoclonal.
The antibody may be conjugated to a functional moiety such as a drug, detectable moiety or a solid support.
Also within the scope of the invention are compositions comprising two or more antibodies which bind different epitopes of ApoE-CTD. The antibodies in the composition may bind overlapping epitopes. Antibodies that bind overlapping epitopes competitively inhibit the binding of each other to ApoE-CTD.
The antibody is preferably monospecific, e.g. a monoclonal antibody, or antigen-binding fragment thereof. Bispecific and multivalent antibodies are also provided, which bispecific or multivalent antibodies bind to two or more different epitopes of ApoE-CTD.
An antibody of the invention may be joined to a binding moiety such as biotin. For example, an antibody, preferably an IgG, may be biotinylated by incubation with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. A biotinylated IgG preferably comprises from 1 to 5 such as 2, 3 or 4 biotin groups.
An antibody of the invention may be in substantially isolated form. They may be mixed with carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95%, 98% or 99%, of the polypeptides or dry mass of the preparation.
B. Methods For Identifying Antibodies
The invention also provides a method for identifying an antibody according to the invention. The method typically comprises identifying an antibody that binds to a polypeptide having the amino acid sequence as shown in SEQ ID NO: 1 or the amino acid sequence of a part thereof and that binds to human plaques. Either or both binding assays may be carried out in the presence of VLDL. The methods generally comprise providing a display library and screening the library to identify a member that encodes an antibody that binds to ApoE-CTD or a fragment thereof and/or to human plaques, preferably in the presence of VLDL. A display library is a collection of entities; each entity includes an accessible antibody component and a recoverable component that encodes or identifies the antibody component. The antibody component can be of any length, e.g., from three amino acids to over 300 amino acids for example 30, 100 or 200 amino acids and is typically an antibody fragment, preferably a Fab fragment. In a selection, the antibody component of each member of the library is probed with ApoE-CTD and if the antibody component binds to ApoE-CTD or fragment thereof, the display library member is identified, typically by retention on a support. Display library members that bind ApoE-CTD may also typically tested for binding to ApoE-NTD (negative selection).
Retained display library members are recovered from the support and analysed. The analysis can include amplification and a subsequent selection under similar or dissimilar conditions. For example, positive and negative selections can be alternated. The analysis can also include determining the amino acid sequence of the antibody component and purification of the antibody component for detailed characterisation.
A variety of formats can be used for display libraries and any suitable format may be used in a method of the invention. Preferred formats are phage display and cell-based display such as yeast display.
In phage display, the candidate antibodies are typically covalently linked to bacteriophage coat protein. The linkage results from translation of a nucleic acid encoding the candidate antibodies fused to the coat protein. The linkage can include a flexible peptide linker, a protease site, or an amino acid incorporated as a result of suppression of a stop codon. Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/02809; WO 90/09690; de Haard et al. (1999) J. Biol. Chem. 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4: 1-20; Hoogenboom et al. (2000) Immunol. Today 2:371-8; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377; Rebar et al. (1996) Methods Enzymol. 267:129-49; Hoogenboom et al. (1991) Nuc. Acids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and Lee et al. (2003) Trends In Biotechnology 21: 45-52.
Phage display systems have been developed for filamentous phage (phage fl, fd and M13) as well as other bacteriophage (e.g., T7 Bacteriophage and lambdoid phages; see, e.g., Santini (1998) J. Mol. Biol. 282:125-135; Rosenberg et al. (1996) Innovations 6:1-6; Houshmet et al. (1999) Anal. Biochemn. 268:363-370). The filamentous phage display systems typically use fusions to a minor coat protein, such as gene III protein, and gene VIII protein, a major coat protein, but fusions to other coat proteins such as gene VI protein, gene VII protein, gene IX protein, or domains thereof can also been used (see, e.g., WO 00/71694). In a preferred embodiment, the fusion is to a domain of the gene III protein, e.g., the anchor domain or “stump,” (see, e.g., U.S. Pat. No. 5,658,727 for a description of the gene III protein anchor domain).
The valency of the candidate polypeptides can also be controlled. Cloning of the sequence encoding the polypeptide component into the complete phage genome results in multivariant display since all replicates of the gene III protein are fused to the polypeptide component. For reduced valency, a phagemid system can be utilised. In this system, the nucleic acid encoding the polypeptide component fused to gene III is provided on a plasmid, typically of length less than 700 nucleotides. The plasmid includes a phage origin of replication so that the plasmid is incorporated into bacteriophage particles when bacterial cells bearing the plasmid are infected with helper phage, e.g., M13K07. The helper phage provides an intact copy of gene III and other phage genes required for phage replication and assembly. The helper phage has a defective origin such that the helper phage genome is not efficiently incorporated into phage particles relative to the plasmid that has a wild type origin.
Bacteriophage displaying the candidate antibodies can be grown and harvested using standard phage preparatory methods, e.g., PEG precipitation from growth media.
After selection of individual display phages, the nucleic acid encoding the selected candidate antibodies can be obtained by infecting cells using the selected phages. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced.
In a screening procedure to obtain ApoE-CTD binders according to this invention, a display library is contacted with and allowed to bind a target ApoE-CTD molecule, usually immobilised on a solid support. Non-binders are separated from binders. In various ways, the bound phage are liberated from the ApoE-CTD, collected and amplified. Since the phage can be amplified through infection of bacterial cells, even a few binding phage are sufficient to reveal the gene sequence that encodes a binding entity. Using these techniques it is possible to recover a binding phage that is about 1 in 20 million in the population. One or more libraries, displaying 10-20 million or more potential binding polypeptides each, can be rapidly screened to find high-affinity ApoE-CTD binders. When the selection process works, the diversity of the population falls with each round until only good binders remain, i.e., the process converges. Typically, a phage display library will contain several closely related binders (10 to 50 binders out of 10 million). Indications of convergence include increased binding (measured by phage titers) and recovery of closely related sequences.
In a cell-display library the candidate antibodies are displayed on the surface of a cell, e.g., a eukaryotic or prokaryotic cell. Exemplary prokaryotic cells include E. coli cells, B. subtilis cells and spores (see, e.g., Lu et al. (1995) Biotechnology 13:366). Exemplary eukaryotic cells include yeast (e.g., Saccharomyces cerevisiae, Schizosaccharmyces pombe, Hanseulla, or Pichia pastoris). Yeast surface display is described in, for example, Boder and Wittrup (1997) Nature Biotech. 15:553-557. A yeast display system that may be used to display immunoglobulin proteins such as Fab fragments, and yeast mating may be used to generate combinations of heavy and light chains.
Yeast display has clear advantages over phage display in the application of affinity maturation of anti-ApoE-CTD antibodies. The most important advantage is that FACS selection may be used to quantitatively sort each yeast cell for its antigen binding. It is also possible to perform normalized selection so that variations in display level can be corrected, thus avoiding selection on the basis of avidity. This is particularly important when using a multivalent target antigen.
The display library may be a ribosome display library. In a ribosome display library mRNA and the candidate antibody encoded by the RNA can be physically associated by stabilising ribosomes that are translating the mRNA and have the nascent polypeptide still attached. Typically, high divalent Mg2+ concentrations and low temperature are used. See, e.g., Mattheakis at al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes at al. (2000) Nature Biotech. 18:1287-92; Hans at al. (2000) Methods Enzymol. 328-404-30 and Schaffitzel et al. (1999) J. Immunol. Methods 231:119-35.
Another display library format utilises peptide-nucleic acid fusions. Polypeptide-nucleic acid fusions can be generated by the in vitro translation of mRNA that include a covalently attached puromycin group, e.g., as described in Roberts and Szostak (1997) Proc. Acad. Sci. USA 94:12297-12302, and U.S. Pat. No. 6,207,446. The mRNA can then be reverse transcribed into DNA and crosslinked to the polypeptide.
Another display format that may be used is a non-biological display in which the antibody component is attached to a non-nucleic acid tag that identifies the antibody. For example, the tag can be a chemical tag attached to a bead that displays the antibody or a radiofrequency tag (See, e.g., U.S. Pat. No. 5,874,214).
A parental binding domain is selected to serve as a structural template for the candidate antibodies. The binding domain may be a naturally occurring or synthetic protein, or a region or domain of a protein such as an immunoglobulin. The parental binding domain may be selected based on knowledge of a known interaction between the parental binding domain and ApoE-CTD but, but this is not critical. In fact, it is not essential that the parental binding domain have any affinity for ApoE-CTD at all: its purpose is to provide a structure from which a library can be generated, which library will include one or more candidate antibodies that bind specifically to ApoE-CTD.
The candidate antibodies may be Fab fragments, single chain Fv molecules (scFV), single domain antibodies, camelid antibodies and camelized antibodies.
In a preferred embodiment, the parental binding domain comprises an immunoglobulin domain with antigen-binding activity, such as scFv, Fab or IgG. A typical display library displays candidate polypeptides that include a VH domain and a VL domain. As in the case of the Fab and other formats, the displayed antibody can include a constant region as part of a light or heavy chain. In one embodiment, each chain includes one constant region, e.g. as in the case of a Fab. In other embodiments, additional constant regions are displayed.
Display libraries are particularly useful, for example for identifying human or “humanised” antibodies that recognise human antigens. The in vitro display selection process surmounts the inability of a normal human immune system to generate antibodies against self-antigens.
Antibody libraries can be constructed by a number of processes (see, e.g. de Haard et al (1999) J. Biol. Chem. 274:18218-30; Hoogenboom et al (1998) Immunotechnology 4:1-20, and Hoogenboom et al (2000) Immunol. Today 21:371-8). Further, elements of each process can be combined with those of other processes. The processes can be used such that variation is introduced into a single immunoglobulin domain (e.g. VH or VL) or into multiple immunoglobulin domains (e.g. VH and VL). The variation can be introduced into an immunoglobulin variable domain, e.g. in the region of one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4, referring to such regions of either and/or both of heavy and light chain variable domains. In one embodiment, variation is introduced into all three CDRs of a given variable domain. In another preferred embodiment, the variation is introduced into CDR1 and CDR2, e.g. of a heavy chain variable domain. Any combination is feasible.
In a preferred embodiment the parental domain comprises the CDR3 sequence shown in any one of SEQ ID NOS: 23, 26, 29, 32, 35, 38, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 95, 113, 119 and 125. Amino acid substitutions at one or both of positions 2 and 3 of SEQ ID NO: 23 or 26 are preferred variations in candidate antibodies. Examples of variant VH-CDR3 sequences generated by antibody spiking are identified in Tables 38, 39, 40, 41 and 42. Preferred CDR3 sequences are shown in SEQ ID NOS: 207, 209, 210, 320, 322, 323 and 373. Consensus sequences for preferred CDR3 sequences are shown in SEQ ID NOS: 512, 513, 514, 515, 516, 517 and 20.
Examples of variant VL-CDR3 sequences generated by light chain shuffling are also shown in Tables 38, 39, 40, 41 and 42. Preferred VL-CDR3 sequences are shown in SEQ ID NOS: 35, 269, 275, 268, 341 and 378.
The parental domain preferably also comprises the other components of the VH chain, and optionally a VL chain, or the other components of the VL chain, and optionally a VH chain.
A second preferred parental domain comprises a CDR1 and/or CDR2 domain with the sequence shown in any one of SEQ ID NOS: 21, 24, 27, 30, 33, 36, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 93, 111, 117 and 123 or in SEQ ID NOS: 22, 25, 28, 31, 34, 37, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 94, 112, 118 and 124. The candidate polypeptides may be generated by DNA shuffling the CDR1 and/or CDR2 domains. Examples of variant VL-CDR1 and VL-CDR2 sequences are identified in Tables 38, 39, 40, 41 and 42. Preferred CDR1 sequences are shown in SEQ ID NOS: 33, 219, 226, 218, 93, 325, 326, 391 and 394. Preferred CDR2 sequences are shown in SEQ ID NOS: 34, 247, 252, 333, 334, 382 and 386.
A third preferred parental domain comprises the VL sequence shown in any one of SEQ ID NOS: 42, 43, 44, 151, 157, 159 and 161. Candidate polypeptides are typically generated by DNA shuffling of the entire VL sequence. Examples of shuffled light chain sequences are identified in Tables 38, 39, 40, 41 and 42. Preferred shuffled light chain sequences are shown in SEQ ID NOS: 43, 295, 294, 304, 347, 348, 357, 362, 406 and 418.
In one process, antibody libraries are constructed by inserting diverse oligonucleotides that encode CDRs into the corresponding regions of the nucleic acid. The oligonucleotides can be synthesized using monomeric nucleotides or trinucleotides. For example, Knappik et al ((2000) J Mol. Biol. 296:57-86) describes a method for constructing CDR encoding oligonucleotides using trinucleotide synthesis and a template with engineered restriction sites for accepting the oligonucleotides.
In another process, an animal, e.g. a rodent, is immunised with the ApoE-CTD. The animal is optionally boosted with the antigen to further stimulate the response. Then spleen cells are isolated from the animal, and nucleic acid encoding VH and/or VL domains is amplified and cloned for expression in the display library.
In yet another process, antibody libraries are constructed from nucleic acid amplified from naïve germline immunoglobulin genes. The amplified nucleic acid includes nucleic acid encoding the VH and/or VL domain. Sources of immunoglobulin-encoding nucleic acids are described below. Amplification can include PCR, e.g. with primers that anneal to the conserved constant region, or another amplification method.
Nucleic acid encoding immunoglobulin domains can be obtained from the immune cells of, e.g. a human, a primate, mouse, rabbit, camel or rodent. In one example, the cells are selected for a particular property. B cells at various stages of maturity can be selected. In another example, the B cells are naïve.
In one embodiment, fluorescent-activated cell sorting (FACS) is used to sort B cells that express surface-bound IgM, IgD or IgG molecules. Further, B cells expressing different isotypes of IgG can be isolated. In another preferred embodiment, the B or T cell is cultured in vitro. The cells can be stimulated in vitro, e.g. by culturing with feeder cells or by adding mitogens or other modulatory reagents, such as antibodies to CD40, CD40 ligand or CD20, phorbol myristate acetate, bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin or pokeweed mitogen.
In still another embodiment, the cells are isolated from a subject that has an immunological disorder, e.g. systemic lupus erythematosus (SLE), rheumatoid arthritis, vasculitis, Sjogren syndrome, systemic sclerosis or anti-phospholipid syndrome. The subject can be a human or an animal, e.g. an animal model for the human disease, or an animal having an analogous disorder. In yet another embodiment, the cells are isolated from a transgenic non-human animal that includes a human immunoglobulin locus.
In one preferred embodiment, the cells have activated a program of somatic hypermutation. Cells can be stimulated to undergo somatic mutagenesis of immunoglobulin genes, for example, by treatment with anti-immunoglobulin, anti-CD40 and anti-CD38 antibodies (see, e.g. Bergthorsdottir et al (2001) J. Immunol 166:2228). In another embodiment, the cells are naïve.
The nucleic acid encoding an immunoglobulin variable domain can be isolated from a natural repertoire by the following exemplary method. First, RNA is isolated from the immune cell. Full length (i.e. capped) mRNAs are separated (e.g. by degrading uncapped RNAs with calf intestinal phosphatase). The cap is then removed with tobacco acid pyrophosphatase and reverse transcription is used to produce the cDNAs.
The reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g. de Haard et al (1999) J. Biol. Chem. 274:18218-30. The primer binding region can be constant among different immunoglobulins, e.g. in order to reverse transcribe different isotypes of immunoglobulin. The primer binding region can also be specific to a particular isotype of immunoglobulin. Typically, the primer is specific for a region that is 3′ to a sequence encoding at least one CDR. In another embodiment, poly-dT primers may be used (and may be preferred for the heavy-chain genes).
A synthetic sequence can be ligated to the 3′ end of the reverse transcribed strand. The synthetic sequence can be used as a primer binding site for binding of the forward primer during PCR amplification after reverse transcription. The use of the synthetic sequence can obviate the need to use a pool of different forward primers to fully capture the available diversity.
The variable domain-encoding gene is then amplified, e.g. using one or more rounds. If multiple rounds are used, nested primers can be used for increased fidelity. The amplified nucleic acid is then cloned into a display library vector.
Any method for amplifying nucleic acid sequences may be used for amplification. Methods that maximise, and do not bias, diversity are preferred. Suitable techniques for nucleic acid amplification include the polymerase chain reaction (PCR), transcription-based methods that utilise RNA synthesis by RNA polymerases to amplify nucleic acid (Sarker et al (1989) Science 244:331-34), NASBA (U.S. Pat. Nos. 5,130,238; 5,409,818; and 5,554,517) which utilises cycles of transcription, reverse-transcription and RnaseH-based degradation to amplify a DNA sample, rolling circle amplification (RCA; U.S. Pat. No. 6,143,495) and strand displacement amplification (SDA; U.S. Pat. No. 5,624,825).
After a first set of binding antibodies is identified, the sequence information can be used to design other libraries biased for members having additional desired properties, e.g., discrimination between ApoE-CTD and full-length ApoE, preferably VLDL-associated ApoE. Such techniques make it possible not only to screen a large number of potential binding antibodies but also make it practical to repeat the binding/elution cycles and to build secondary, biased libraries for screening analog-displaying packages that meet initial criteria Using these techniques, a biased library may be screened to reveal members that bind tightly (i.e., with high affinity) under the screening conditions.
Thus, in one preferred embodiment, display library technology may be used in an iterative mode. A first display library is used to identify one or more antibodies that bind ApoE-CTD and/or human plaques. These identified antibodies are then varied using a mutagenesis method to form a second display library. Higher affinity polypeptides are then selected from the second library, e.g., by using higher stringency or more competitive binding and washing conditions.
In affinity maturation protocols, the variation is preferably generated by amino acid substitutions but may also result from deletion or addition of amino acids.
The amino acid substitutions may be those which are expected to alter the binding properties of the domain without significantly altering its structure, at least for most substitutions. It is preferred that the amino acid positions that are selected for variation (variable amino acid positions) will be surface amino acid positions, that is, positions in the amino acid sequence of the domains which, when the domain is in its most stable conformation, appear on the outer surface of the domain (i.e., the surface exposed to solution). Most preferably the amino acid positions to be varied will be adjacent or close together, so as to maximise the effect of substitutions. In addition, extra amino acids can be added into the structure of the parental binding domain.
In some implementations, the mutagenesis is targeted to regions known or likely to be at the binding interface. Mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs. Mutagenesis can also be limited to one or a few of the CDRs, e.g., to make precise step-wise improvements.
Effective affinity maturation requires 4 components (i) the rediversification of lead antibody genes (ii) display on either phage or yeast (iii) affinity selection (iv) screening of clones for improved affinity.
Alignment of Fabs showing the required binding properties, for example using a BLAST algorithm (e.g. Karlin and Altschul (1993) PNAS USA 90: 5873-5787) may be used to identify conserved residues in the CDR domains. Sequence similarity amongst the CDR loops may allow a prediction of the direct involvement of any amino acid in antibody affinity or specificity.
For example, the VH-CDR3 loops of antibodies 807A-M0028-B02 and 807A-M0027-E11 (SEQ ID NOS: 23 and 26) show striking similarity and show consensus over 4/6 amino acids (SEQ ID NO: 20). This suggests that the VH-CDR3 plays a role in antibody affinity and specificity.
An optimal antibody mutagenesis strategy introduces a minimal number of mutations at functionally relevant positions. This is achieved by both targeted and non-targeted mutagenesis procedures. Non-targeted mutagenesis procedures include chain shuffling which introduces large block changes in antibodies by rediversifying the whole VL gene or the VH CDR1-2 fragment. Typically, the VH-CDR3 loop is left untouched, as it may make significant contributions to binding affinity and specificity. Examples of chain shuffling are described in the following documents: Marks et al., (1992) Nature Biotech 10: 779-783, Schier et al., (1996) J. Mol. Biol. 255, 28-43, Park et al., (2000) BBRC. 275. 553-557 and Chames et al., (2002) J. Immunol. 1110-1118. Although chain shuffling is a well-validated technique (particularly for antibodies with a low starting affinity) a possible disadvantage is that by making such large block changes in the antibody molecule that there may be an increased chance of disrupting multiple favourable contacts. However this could be compensated by the loss of unfavourable contacts or the generation of new contacts.
Other exemplary non-targeted mutagenesis techniques include: error-prone PCR (Leung et al. (1989) Technique 1:11-15), recombination, DNA shuffling using random cleavage (Stemmer (1994) Nature 389-391; termed “nucleic acid shuffling”), RACHITTTM (Coco at al. (2001) Nature Biotech. 19:354), site-directed mutagenesis (Zooler et al. (1987) Nucl. Acids Res. 10:6487-6504), cassette mutagenesis (Reidhaar-Olson (1991) Methods Enzymol. 208:564-586) and incorporation of degenerate oligonucleotides (Griffiths at al. (1994) EMBO J. 13:3245).
Targeted mutagenesis procedures include hot spot mutagenesis, parsimonious mutagenesis, saturation mutagenesis, domain randomisation and domain walking. CDR mutagenesis can be done in a stepwise manner that is target CDR1, select an optimised loop and subsequently target CDR2 etc. The single most naturally diverse loop is the VH-CDR3 and it is generally accepted that as this loop is situated centrally in the antibody combining site that this is a critical determinant of antibody specificity and affinity. This means that there is a strong case for specifically targeting this loop.
In one example of iterative selection, the methods described herein are used to first identify an antibody from a display library that binds ApoE-CTD with at least a minimal binding specificity for a target or a minimal activity, e.g., an equilibrium dissociation constant for binding of greater than 1 nM, 10 nM, or 100 nM, and which binds human plaques and or/which retains binding activity in the presence of VLDL-associated ApoE. The nucleic acid sequence encoding the initial identified antibody is used as a template nucleic acid for the introduction of variations, e.g., to identify a second polypeptide that has enhanced properties (e.g., binding affinity, kinetics, or stability) relative to the initial antibody.
Antibodies according to the present invention may be isolated using display technology, in a manner to identify ApoE-CTD binding antibodies exhibiting particular preselected properties of binding and release. According to this methodology, two solution conditions may be preselected, i.e., binding conditions and release conditions. The binding conditions are a set of solution conditions under which is desired that a discovered antibody will bind the target ApoE-CTD; the release conditions are a set of solution conditions under which it is desired that a discovered antibody will not bind the ApoE-CTD (i.e. will dissociate from ApoE-CTD). The two conditions may be selected to satisfy any criterion of the practitioner, such as ease of attaining the conditions, compatibility with other purification steps, lowered cost of switching between conditions compared to other affinity media, etc. Preferably, the two solution conditions are selected so as not to adversely affect the stability or activity of the target protein ApoE-CTD and so as to differ significantly with respect to at least one solution parameter. For example, in conducting the screening for suitable binding peptides described herein, binders are selected that dissociated from the target in the presence of an ethylene glycol-containing buffer, or conditions of lowered pH (i.e. pH 2), or combinations of those conditions, which differ from the conditions employed for binding. Another parameter that can be advantageously varied is the concentration of a salt, for example NaCl, in the binding and elution buffers.
An antibody which binds to ApoE-CTD typically has a minimal binding specificity for ApoE-CTD, for example an equilibrium constant for binding of greater than 1 nM, 10 nM or 100 nM.
Since a slow dissociation rate can be predictive of high affinity, particularly with respect to interactions between antibodies and their targets, methods of off-rate selection can be used to isolate antibodies with a desired kinetic dissociation rate (i.e., reduced) for a binding interaction to an ApoE-CTD.
To select for slow dissociating antibodies from a display library, the library is contacted to an immobilised target, preferably ApoE-CTD. The immobilised target is then washed with a first solution that removes non-specifically or weakly bound antibodies. Then the immobilised target is eluted with a second solution that includes a saturation amount of free target, i.e., replicates of the target that are not attached to the particle. The free target binds to antibodies that dissociate from the target. Rebinding is effectively prevented by the saturating amount of free target relative to the much lower concentration of immobilised target.
The second solution can have solution conditions that are substantially physiological or that are stringent. Typically, the solution conditions of the second solution are identical to the solution conditions of the first solution. Fractions of the second solution are collected in temporal order to distinguish early from late fractions. Later fractions include biomolecules that dissociate at a slower rate from the target than biomolecules in the early fractions.
Further, it is also possible to recover display library members that remain bound to the target even after extended incubation. These can either be dissociated using chaotropic conditions or can be amplified while attached to the target. For example, phage bound to the target can be contacted to bacterial cells.
The ApoE-CTD used in a method of the invention may be in any suitable form. ApoE-CTD typically has the amino acid sequence set out in SEQ ID NO. 1 or the amino acid sequence of a fragment thereof. The fragment of ApoE-CTD is typically at least three amino acids in length, preferably at least five, six, seven or eight amino acids in length and more preferably at least 10, 12 or 16 amino acids in length. Examples of suitable fragments are set out in SEQ ID NOS: 2 to 19. Preferred fragments are those having a sequence shown in any one of SEQ ID NOs: 2, 5, 7, 9, 10, 12, 13, 14, 15, 16 and 17. One or more ApoE-CTD peptide may be used in a screening assay of the invention.
The ApoE-CTD polypeptides are generally produced by recombinant means. Urea-denatured ApoE-CTD which has been recombinantly or naturally produced may be used in a method of the invention. Candidate polypeptides may additionally or alternatively be screened for binding to CTD in a polymeric form (ApoE-CTD binds to fibrils). Binding to a complex of ApoE-CTD and AD may also be monitored.
ApoE-CTD may be cleaved from recombinant or naturally occurring ApoE, for example by the action of thrombin.
The ApoE-CTD polypeptide or peptide may be immobilised on a support. Typically immobilisation is achieved by tagging or biotinylating the polypeptide for capture onto a surface. For example, the ApoE-CTD may comprise an S—S biotin group for attachment to streptavadin (for example on streptavadin-coated magnetic beads). Alternatively the ApoE-CTD may comprise a cysteine residue for coupling to a BSA carrier for immobilisation (for example on plastic). In this way a “CTD-coated chip” may be produced. Binding of candidate polypeptide to a CTD-coated chip may be analysed by BIACORE analysis.
Display library members may also be screened for binding to human plaques.
The display library screening methods described herein preferably include a selection or screening process that discards display library members that bind to a non-target molecule. Examples of non-target molecules include: streptavidin and (ii) ApoE-NTD.
In one implementation, a so-called “negative selection” step is used to discriminate between the target and related non-target molecule and a related, but distinct non-target molecule. The display library or a pool thereof is contacted to the non-target molecule. Members of the sample that do not bind the non-target are collected and used in subsequent selections for binding to the target molecule or even for subsequent negative selections. The negative selection step can be prior to or after selecting library members that bind to the target molecule.
In another implementation, a screening step is used. After display library members are isolated for binding to the target molecule, each isolated library member is tested for its ability to bind to a non-target molecule (e.g., a non-target listed above). For example, a high-throughput ELISA screen can be used to obtain this data. The ELISA screen can also be used to obtain quantitative data for binding of each library member to the target. The non-target and target binding data are compared (e.g. using a computer and software) to identify library members that specifically bind to the target MHC-peptide complex.
An antibody or antibody fragment of the invention may bind to ApoE in the presence of VLDL or other lipoprotein particles. An antibody of the invention typically binds to ApoE with a minimal binding specificity for ApoE-CTD, e.g. an equilibrium constant for binding of greater than 1 nM or 100 nM in the presence of VLDL. The VLDL may be present in any suitable form. For example human plasma may be added to the binding assay. Up to 50% human plasma may be added to the assay, for example up to 10%, up to 20%, up to 30% or up to 40% human plasma may be included.
The candidate polypeptides may also be screened for binding to ApoE-NTD. ApoE-NTD may be produced recombinantly or may be cleaved from recombinant or naturally occurring ApoE, for example by the action of thrombin.
In one embodiment, the candidate polypeptides may be screened for binding to astrocytes. It is preferred but not essential, that the selected polypeptides do not bind to astrocytes or bind with a much lower affinity to astrocytes than to ApoE-CTD, for example a two-fold, five-fold, 10-fold, 20-fold or 50-fold lower affinity.
After selecting candidate display library members that bind to ApoE-CTD, each candidate display library member may be further analysed, e.g. to further characterise its binding properties for the target. Each candidate display library member can be subjected to one or more secondary screening assays. The assay can be for a binding property, a catalytic property, a physiological property (e.g. cytotoxicity, renal clearance, immunogenicity), a structural property (e.g. stability, conformation, oligomerisation state) or another functional property. The same assay can be used repeatedly, but with varying conditions, e.g. to determine pH, ionic or thermal sensitivities.
As appropriate, the assays can use the display library member directly, a recombinant antibody produced from the nucleic acid encoding a displayed antibody, or a synthetic antibody synthesised based on the sequence of a displayed antibody. The assays preferably comprise determining whether or not an antibody that binds ApoE-CTD also binds to human plaques, or whether it binds to ApoE-CTD in the presence of VLDL. Exemplary assays for binding properties include ELISA, homogeneous binding assays such as fluorescence resonance energy transfer (FRET) and alpha-screen, surface plasmon resonance (SPR), protein assays and cellular assays.
Antibodies encoded by a display library can also be screened for a binding property using an ELISA. For example, each candidate antibody that binds ApoE-CTD is brought into contact with a microtitre plate whose bottom surface has been coated with ApoE-CTD, VLDL or ApoE-NTD. The plate is washed with buffer to remove non-specifically bound polypeptides. Then the amount of the polypeptide bound to the plate is determined by probing the plate with an antibody that can recognise the polypeptide, e.g. a tag or constant portion of the polypeptide. The antibody is linked to an enzyme such as alkaline phosphatase, which produces a colorimetric product when appropriate substrates are provided. The polypeptide can be purified from cells or assayed in a display library format, e.g. as a fusion to a filamentous bacteriophage coat. In another version of the ELISA, each polypeptide of a diversity strand library is used to coat a different well of a microtitre plate. The ELISA then proceeds using a constant target molecule to query each well. A polypeptide specifically binds ApoE-CTD in an ELISA if it displays at least 2× background on ApoE-CTD but less than 1× background on negative control proteins such as ApoE-NTD or streptavidin.
A homogeneous binding assay is an assay in which the binding interaction of candidate antibody with a target can be analysed after all components of the assay are added without additional fluid manipulations being required. For example, fluorescence resonance energy transfer (FRET) can be used as a homogenous assay (see, for example, Lakowicz et al, U.S. Pat. No. 5,631,169). Another example of a homogenous assay is Alpha Screen (Packard Bioscience, Meriden, Conn., USA).
The homogenous assays can be performed while the candidate polypeptide is attached to the display library vehicle, e.g. a bacteriophage.
The binding interaction of a molecule isolated from a display library and a target can be analysed using Surface Plasmon Resonance (SPR). SPR or Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labelling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in Szabo et al (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line resources provided by BIAcore International AB (Uppsala, Sweden).
Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (KD), and kinetic parameters, including Kon and Koff, for the binding of a biomolecule to a target. Such data can be used to compare different biomolecules. For example, proteins encoded by nucleic acid selected from a library of diversity strands can be compared to identify individuals that have high affinity for the target or that have a slow Koff. This information can also be used to develop structure-activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of matured versions of a parent protein can be compared to the parameters of the parent protein. Variant amino acids at given positions can be identified that correlate with particular binding parameters, e.g. high affinity and slow Koff. This information can be combined with structural modelling (e.g. using homology modelling, energy minimisation or structure determination by crystallography or NMR). As a result, an understanding of the physical interaction between the protein and its target can be formulated and used to guide other design processes.
Antibodies identified from the display library can be immobilised on a solid support, for example, on a bead or an array. For a protein array, each of the polypeptides is immobilised at a unique address on a support. Typically, the address is a two-dimensional address. Protein arrays are described below (see, e.g. “Diagnostics”).
A candidate antibody identified as binding to ApoE-CTD can be screened by transforming vector nucleic acid sequences that encode the antibody into a host cell such that antibodies are produced within the cell, secreted from the cell, or attached to the cell surface. The cells can be screened for antibodies that bind to ApoE-CTD, for example by detecting a change in a cellular phenotype or a cell-mediated activity. For example, the activity may be cell or complement-mediated cytotoxicity.
In another embodiment, the library of cells is in the form of a cellular array. The cellular array can likewise be screened for any appropriate detectable activity.
C. Producing an Antibody
Standard recombinant nucleic acid methods can be used to express an antibody of the invention. Generally, a nucleic acid sequence encoding the antibody is cloned into a nucleic acid expression vector. Of course, if the antibody includes multiple polypeptide chains, each chain must be cloned into an expression vector, e.g. the same or different vectors, that are expressed in the same or different cells. If the antibody fragment is sufficiently small, i.e. has less than 50 amino acids, it can be synthesised using automated organic synthetic methods. Methods for producing antibodies are also provided below.
The expression vector for expressing the antibody ligand can include, in addition to the segment encoding the polypeptide ligand or fragment thereof, regulatory sequences, including for example, a promoter, operably linked to the nucleic acid(s) of interest. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia). One preferred class of preferred libraries is the display library, which is described below.
Methods well known to those skilled in the art can be used to construct vectors containing an antibody of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, NY (2001) and Ausubel et al, Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley Interscience, NY (1989)). Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI lacZ, T3, T7, gpt, lambda P and trc. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, mouse metallothionein-I and various art-known tissue specific promoters.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g. the ampicillin resistance gene of E. coli and S. cerevisiae auxotrophic markers (such as URA3, LEI2, HIS3 and TRPl genes), and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase or heat shock proteins, among others. The polynucleotide of the invention is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, a nucleic acid of the invention can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g. stabilisation or simplified purification of expressed recombinant product. Useful expression-vectors for bacteria are constructed by inserting a polynucleotide of the invention together with suitable translation initiation and termination signals, optionally in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but non-limiting example, useful expression vectors for bacteria can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega, Madison, Wis., USA).
The present invention further provides host cells containing the vectors of the present invention, wherein the nucleic acid has been introduced into the host cell using known transformation, transfection or infection methods. For example, the host cells can include members of a library constructed from the diversity strand. The host cell can be a eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected, for example by calcium phosphate transfection, DEAE, dextran mediated transfection or electroporation (Davis, L. et al, Basic Methods in Molecular Biology (1986)).
Any host/vector system can be used to identify one or more of the target elements of the present invention. These include, but are not limited to, eukaryotic hosts such as HeLa cells, CV-1 cells, COS cells, Sf9 cells and HEK293T cells as well as prokaryotic hosts such as E. coli and B. subtilis. The most preferred cells are those which do not normally express the particular reporter polypeptide or protein or which expresses the reporter polypeptide or protein at low natural level.
The host of the present invention may also be a yeast or other fungi. In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol, 2, Ed. Ausubel et al, Greene Publish. Assoc. & Wiley Interscience, Ch. 13 (1988); Grant et al (1987) “expression and Secretion Vectors for Yeast”, Methods Enzymol. 153:516-544 (1987); and The Molecular Biology of the Yeast Saccharomyces, Eds. Strathem et al, Cold Spring Harbor Press, Vols. I and II (1982).
The host of the invention may also be a prokaryotic cell such as E. coli, other enterobacteriaceae such as Serratia marescans, bacilli, various pseudomonads or other prokaryotes which can be transformed, transfected and/or infected.
The present invention further provides host cells genetically engineered to contain the antibodies of the invention. For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods. The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the antibodies in the cell.
The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAB, dextran mediated transfection or electroporation (David, L. et al, (1986) Basic Methods in Molecular Biology). The host cells containing one of the polynucleotides of the invention can be used in a conventional manner to produce the gene product encoded by the isolated fragment (in the case of an ORF).
Any on suitable host/vector system can be used to express one or more of the diversity antibodies of the present invention. Various mammalian cell culture systems can also be employed to express recombinant antibodies.
Antibodies, e.g. Fabs, can be produced in bacterial cells, e.g. E. coli cells. For example, if the Fab is encoded by sequences in a phage display vector that includes a suppressible stop codon between the display entity and a bacteriophage protein (or fragment thereof), the vector nucleic acid can be shuffled into a bacterial cell that cannot suppress a stop codon. In this case, the Fab is not fused to the gene ImI protein and is secreted into the media Antibodies can also be produced in eukaryotic cells. In one embodiment, the antibodies (e.g. scFvs) are expressed in a yeast cell such as Pichia (see, e.g. Powers et al (2001) J. Immunol. Methods. 251:123-35), Hanseula or Saccharomyces. 
In one preferred embodiment, antibodies are produced in mammalian cells. Preferred mammalian host cells for expressing the clone antibodies or antigen-binding fragments thereof include Chinese Hamster Ovary (CHO cells) (including dhfr− CHO cells, described in Urlaub and Chasin ((1980) Proc. Natl. Acad. Sci. USA 77:4216-4220), used with a DHFR selectable marker, e.g. as described in Kaufman and Sharp ((1982) Mol. Biol. 159:601-621), lymphocytic cell lines, e.g. NS0 myeloma cells and SP2 cells, COS cells and a cell from a transgenic animal, e.g. a transgenic mammal. For example, the cell is a mammary epithelial cell.
In addition to the nucleic acid sequence encoding the diversified immunoglobulin domain, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g. origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g. U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr− host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
In an exemplary system for recombinant expression of an antibody, or antigen-binding portion thereof, of the invention, a recombinant expression vector encoding both the antibody heavy and the antibody light chain is introduced into dhfr− CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g. derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carried a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G.
For antibodies that include an Fc domain, the antibody production system preferably synthesises antibodies in which the Fc region is glycosylated. For example, the Fc domain of IgG molecule is glycosylated at asparagine 297 in the CH2 domain. This asparagine is the site for modification with biantennary-type oligosaccharides. It has been demonstrated that this glycosylation is required for effector functions mediated by Fc receptors and complement C1q (Burton and Woof (1992) Adv. Immunol. 51:1-84; Jefferis et al (1998) Immunol. Rev. 163:59-76). In one preferred embodiment, the Fc domain is produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297. The Fc domain can also include other eukaryotic post-translational modifications.
Antibodies can also be produced by a transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest. The antibody can be purified from the milk, or for some applications, used directly.
An ApoE-CTD antibody of the invention may be isolated from the display library and its sequence and/or structure may be analysed. The antibody may be produced in any desired quantity using known methods. For example, the antibody may advantageously be produced by a chemical synthesis followed by treatment under oxidising conditions appropriate to obtain the native conformation, i.e., the correct disulfide bond linkages. Synthesis may be carried out by methodologies well known to those skilled in the art (see, Kelley et al., in Genetic Engineering Principles and Methods, (Setlow, J. K., ed.), Plenum Press, NY., (1990) vol. 12, pp. 1-19; Stewart et al., Solid-Phase Peptide Synthesis (1989), W.H. Freeman Co., San Francisco). Polypeptides according to the invention may also be prepared commercially by companies providing polypeptide synthesis as a service (e.g., BACHEM Bioscience, Inc., King of Prussia, Pa.; Quality Controlled Biochemicals, Inc., Hoplinton, Mass.).
D. Diagnostic Methods
Antibodies that bind to ApoE-CTD and identified by the methods described herein and/or detailed herein have in vitro and in vivo diagnostic, therapeutic and prophylactic utilities.
In one aspect, the present invention provides a diagnostic method for detecting the presence ApoE-CTD in vitro (e.g., a biological sample, such as a biopsy or in vivo (e.g., in vivo imaging in a subject).
The method includes: (i) contacting a sample with an antibody of the invention; and (ii) detecting formation of a complex between the antibody and the sample. The method can also include contacting a reference sample (e.g., a control sample) with the antibody, and determining the extent of formation of the complex between the antibody and the sample relative to the same for the reference sample. A change, e.g., a statistically significant change, in the formation of the complex in the sample or subject relative to the control sample or subject can be indicative of the presence of ApoE-CTD in the sample.
Another method includes: (i) administering an antibody of the invention to a subject; and (ii) detecting formation of a complex between the antibody and the subject. The detection step can include determining location or time of formation of the complex.
The antibody ligand can be directly or indirectly labelled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
Complex formation between an antibody of the invention and ApoE-CTD can be detected by measuring or visualizing either the antibody bound to the ApoE-CTD or unbound antibody. Conventional detection assays can be used, e.g., an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry. Further to labelling the antibody, the presence of ApoE-CTD can be assayed in a sample by a competition immunoassay utilising standards labelled with a detectable substance and an unlabelled antibody. In one example of this assay, the biological sample, the labelled standards and the antibody are combined and the amount of labelled standard bound to the unlabeled ligand is determined. The amount of ApoE-CTD in the sample is inversely proportional to the amount of labelled standard bound to antibody.
Fluorophore and chromophore labelled antibodies can be prepared. Since antibodies absorb light having wavelengths up to about 310 nm, the fluorescent moieties should be selected to have substantial absorption at wavelengths above 310 nm and preferably above 400 nm. A variety of suitable fluoresces and chromophores are described by Stryer (1968) Science 162:526 and Brand, L. et al. (1972) Annual Review of Biochemistry 41:843-868. The antibodies can be labelled with fluorescent chromophore groups by conventional procedures such as those disclosed in U.S. Pat. Nos. 3,940,475, 4,289,747 and 4,376,110. One group of fluorescers having a number of the desirable properties described above is the xanthene dyes, which include the fluoresceins and rhodamines. Another group of fluorescent compounds are the naphylamines. Once labelled with a fluorophore or chromophore, the antibody can be used to detect the presence or localisation of the ApoE-CTD in a sample, e.g., using fluorescent microscopy (such as confocal or deconvolution microscopy).
Immunohistochemistry can be performed using the antibodies described herein. For example, the antibody can synthesised with a label (such as a purification or epitope tag), or can be detectably labelled, e.g., by conjugating a label or label-binding group. For example, a chelator can be attached to the antibody. The antibody is then contacted to a histological preparation, e.g., a fixed section of tissue that is on a microscope slide. After an incubation for binding, the preparation is washed to remove unbound antibody. The preparation is then analysed, e.g., using microscopy, to identify if the antibody bound to the preparation.
Of course, the antibody can be unlabelled at the time of binding. After binding and washing, the antibody is labelled in order to render it detectable.
The antibody can also be immobilised on a protein array. The protein array can be used as a diagnostic tool, e.g., to screen medical samples (such as isolated cells, blood, sera, biopsies, and the like). Of course, the protein array can also include other ligands, e.g., that bind to the ApoE-CTD.
Methods of producing polypeptide arrays are described, e.g., in De Wildt et al. (2000) Nature Biotech. 18:989-994; Lueking et al. (1999) Anal. Biochem. 270:103-111; Ge (2000) Nuc. Acids Res. 28:e3; MacBeath and Schreiber (2000) Science 289:1760-1763; WO 01/40803 and WO 99/51773A1. Polypeptides for the array can be spotted at high speed, e.g., using commercially available robotic apparati, e.g., from Genetic MicroSystems and Affymetrix (Santa Clara, Calif., USA) or BioRobotics (Cambridge, UK). The array substrate can be, for example, nitrocellulose, plastic, glass, e.g., surface-modified glass. The array can also include a porous matrix, e.g., acrylamide, agarose or another polymer.
For example, the array can be an array of antibodies, e.g., as described in De Wildt, supra. Cells that produce the polypeptide ligands cm be grown on a filter in an arrayed format. Polypeptide production is induced, and the expressed polypeptides are immobilised to the filter at the location of the cell.
An antibody array can be contacted with a labelled target to determine the extent of binding of the target to each immobilised antibody from the diversity strand library. If the target is unlabeled, a sandwich method can be used, e.g., using a labelled probed, to detect binding of the unlabeled target.
Information about the extent of binding at each address of the array can be stored as a profile, e.g., in a computer database. The antibody array can be produced in replicates and used to compare binding profiles, e.g., of a target and a non-target. Thus, antibody arrays can be used to identify individual members of the diversity strand library that have desired binding properties with respect to one or more molecules.
In still another embodiment, the invention provides a method for detecting the presence of a ApoE-CTD containing plaque in vivo. The method includes (i) administering to a subject (e.g., a patient having Alzheimer's disease or systemic amyloidosis) an antibody of the invention, conjugated to a detectable marker; (ii) exposing the subject to a means for detecting said detectable marker bound to the ApoE-CTD containing plaque. For example, the subject is imaged, e.g., by NMR or other tomographic means.
Examples of labels useful for diagnostic imaging in accordance with the present invention include radiolabels such as 131I, 111In, 123I, 99mTc, 32P, 125I, 3H, 14C, and 188Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography (“PET”) scanner, chemiluminescers such as luciferin and enzymatic markers such as peroxidase or phosphatase. Short-range radiation emitters, such as isotopes detectable by short-range detector probes can also be employed. The polypeptide ligand can be labelled with such reagents using known techniques. Foe example, see Wensel and Meares (1983) Radioimmunoimaging and Radioimmunotherapy, Elsevier, N.Y. for techniques relating to the radiolabel of antibodies and D. Colcher et al. (1986) Methods Enzymol. 121:802-816.
A radiolabel ligand of this invention can also be used for in vitro diagnostic tests. The specific activity of a isotopically-labelled ligand depends upon the half-life, the isotopic purity of the radioactive label, and how the label is incorporated into antibody.
Procedures for labelling polypeptides with the radioactive isotopes (such as 14C, 3H, 35S, 125I, 32P, 131I) are generally known. For example, tritium labelling procedures are described in U.S. Pat. No. 4,302,438. Iodinating, tritium labelling, and 35S labelling procedures, e.g., as adapted for murine monoclonal antibodies, are described, e.g., by Goding, J. W. (Monoclonal Antibodies: Principles And Practice: Production And Application Of Monoclonal Antibodies In Cell Biology, Biochemistry, And Immunology 2nd ed., London, Orlando, Academic Press (1986) polypeptide. 124-126) and the references cited therein. Other procedures for iodinating polypeptides, such as antibodies, are described by Hunter and Greenwood (1962) Nature 144:945, David et al. (1974) Biochemistry 13:1014-1021, and U.S. Pat. Nos. 3,867,517 and 4,376,110. Radiolabelling elements which are useful in imaging include 123I, 131I, 111In, and 99mTc, for example. Procedures for iodinating antibodies are described by Greenwood, F. et al. (1963) Biochem. J. 89:114-123; Marchalonis, J. (1969) Biochem. J. 113:299-305; and Morrison, M. et al. (1971) Immunochemistry 8:289-297. Procedures for 99mTc-labeling are described by Rhodes, B. et al. in Burchiel, S. et al. (eds.), Tumor Imaging: The Radioimmunichemical Detection of Cancer, New York: Masson 111-123 (1982) and the references cited therein. Procedures suitable for 111In-labeling antibodies are described by Hnatowich, D. J. et al. (1983) J. Immun. Methods 65:147-157, Hnatowich, D. et al. (1984) J. Applied Radiation 35:554-557 and Buckley, RG. et al. (1984) F. E. B. S. Lett. 66:202-204.
In the case of a radiolabelled antibody, the antibody is administered to the patient, is localised to the plaque with which the antibody reacts, and is detected or “imaged” in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography. Alternatively, a position emission transaxial tomography scanner, such as designated Pet VI located at Brookhaven National Laboratory, can be used where the radiolabel emits positrons (e.g., 11C, 18F, 15O, and 13N).
Magnetic Resonance Imaging (MRI) uses NMR to visualise internal features of a living subject, and is useful for prognosis, diagnosis, treatment, and surgery. MRI can be used without radioactive tracer compounds for obvious benefit. Some MRI techniques are summarised in published European patent application EP-A-0 502 814. Generally, the differences related to relaxation time constants T1 and T2 of water protons in different environments is used to generate an image. However, these differences can be insufficient to provide sharp high resolution images.
The differences in these relation time constants can be enhanced by contrast agents. Examples of such contrast agents include a number of magnetic agents paramagnetic agents (which primarily alter T1) and ferromagnetic or superaramagnetic (which primarily alter T2 response). Chelates (e.g., EDTA, DTPA and NTS chelates) can be used to attach (and reduce toxicity) of some paramagnetic substances (e.g., Fe+3, Mf+2, Gd+3). Other agents can be in the form of particles, e.g., less than 10 μm to about 10 nM in diameter). Particles can have ferromagnetic, antiferromagnetic or superparamagnetic properties. Particles can include, e.g., magnetic (Fe3O4),—Fe2O3, ferrites and other magnetic mineral compounds of transition elements. Magnetic particles may include one or more magnetic crystals with and without nonmagnetic material. The nonmagnetic material can include synthetic or natural polymers such as sepharose, dextran, dextrin, starch and the like.
Antibodies of the invention can also be labelled with an indicating group containing of the NMR-active 19F atom, or a plurality of such atoms inasmuch as (i) substantially all of naturally abundant fluorine atoms are the 19F isotope and, thus, substantially all fluorine-containing compounds are NMR-active; (ii) any chemically active polyfluorinated compounds such as trifluoracetic anhydride are commercially available at relatively low cost, and (iii) many fluorinated compounds have been found medically acceptable for use in humans such as the perfluorinated polyethers utilised to carry oxygen as hemoglobin replacements. After permitting such time for incubation, a MRI scan is carried out using an apparatus such as one of those described by Pykett (1982) Scientific American 246:78-88.
Also within the scope of the invention are kits comprising an antibody of the invention and instructions for diagnostic use, e.g., the use of the antibody to detect ApoE-CTD, in vitro, e.g., in a sample, e.g., a biopsy from a patient having systemic amyloidosis, or in vivo, e.g., by imaging a subject. The kit can further contain a least one additional reagent, such as a label or additional diagnostic agent. For in vivo use the antibody can be formulated as a pharmaceutical composition.
E. Therapeutic Methods
Polypeptides that bind to ApoE-CTD and identified by the methods described herein and/or detailed herein have therapeutic and prophylactic utilities. For example, these ligands can be administered to cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g. in vivo, to treat, prevent and/or diagnose a variety of disorders such as Alzheimer's disease or systemic amyloidosis.
As used herein, the term “treat” or “treatment” is defined as the application or administration of an anti-ApoE-CTD antibody, alone or in combination with, a second agent to a subject, e.g. a patient, who has a disorder (e.g. a disorder as described herein), a symptom of a disorder or a predisposition toward a disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder.
As used herein, an amount of an anti-ApoE-CTD polypeptide effective to treat a disorder, or a “therapeutically effective amount” refers to an amount of the ligand which is effective, upon single or multiple dose administration to a subject, or in prolonging curing, alleviating, relieving or improving a subject with a disorder as described herein beyond that expected in the absence of such treatment.
As used herein, an amount of an anti-ApoE-CTD polypeptide effective to prevent a disorder, or a “prophylactically effective amount” of the polypeptide refers to an amount of an anti-ApoE-CTD polypeptide, e.g. an anti-ApoE-CTD antibody described herein, which is effective, upon single- or multiple-dose administration to the subject, in preventing or delaying the occurrence of the onset or recurrence of a disorder, e.g. Alzheimer's disease.
The terms “induce”, “inhibit”, “potentiate”, “elevate”, “increase”, “decrease” or the like, e.g. which denote quantitative differences between two states, refer to a difference, e.g. a statistically significant difference, between the two states.
As used herein, the term “subject” is intended to include human and non-human animals. Preferred human animals include a human patient having a disorder characterised by abnormal cell proliferation or cell differentiation. The term “non-human animals” of the invention includes all vertebrates, e.g. non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, sheep, dog, cow, pig, etc.
The term “amyloid disorders” is intended to include, but not limited to, Alzheimer's disease, primary systemic amyloidosis, secondary systemic amyloidosis, senile systemic amyloidosis, familial amyloid polyneuropathy I, familial amyloid polyneuropathy III, familial non-neuropathic amyloidosis, hereditary cerebral amyloid angiopathy, Familial British Dementia (FBD), Haemodialysis-related amyloidosis, Familial amyloidosis (Finnish type), Familial subepithelial corneal amyloid, Type II diabetes, Hereditary renal amyloidosis, Pituitary-gland amyloidosis, Injection localized amyloidosis, Medullary carcinoma of the thyroid, Atrial amyloidosis, Familial Danish Dementia (FDD), and Downs syndrome. Related to amyloid diseases wherein amyloid fibrils are detected, comprise, but is not limited to, Spongiform encephalopathies, Sporadic Creutzfeldt-Jakob disease, Familial Creutzfeldt-Jakob disease, Iatropic prion disorders, Variant Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker Disease (GSS), Kuru, Parkinson's disease, Huntington's disease, Familial amyotrophic lateral sclerosis (ALS), and Chronic obstructive pulmonary disease.
Furthermore, amyloid conditions can be defined as disorders with amyloid deposits in brain, medulla or other organs. An example of such disorders is Alzheimer's disease. Other dementia disorders characterized by amyloid deposits are Spongiform encephalopathies, Sporadic Creutzfeldt-Jakob disease, Familial Creutzfeldt-Jakob disease, Iatropic prion disorders, Variant Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker Disease (GSS), Kuru, Parkinson's disease, Huntington's disease, Familial British Dementia, Familial Danish Dementia, Down syndrome, Primary Systemic amyloidosis, such as Immunoglobulin-light-chain-related amyloidosis, Secondary Systemic amyloidosis, such as Amyloidosis related to amyloid A protein, Familial systemic amyloidosis, such as Familial transthyretin-associated amyloidosis, Familial apolipoprotein A-I associated amyloidosis, Familial gelsolin associated amyloidosis, Familial fibrinogen A α associated amyloidosis, Familial lyzosome amyloidosis, Senile Systemic amyloidosis, Familial amyloid polyneuropathy I, Familial amyloid polyneuropathy III, Familial non-neuropathic amyloidosis, Hereditary cerebral amyloid angiopathy, Haemodialysis-related amyloidosis, Familial amyloidosis, finnish type, Familial subepithelial corneal amyloid, Type II diabetes, Hereditary renal amyloidosis, Pituitary-gland amyloidosis, Injection localized amyloidosis, Medullary carcinoma of the thyroid, Atrial amyloidosis, Chronic obstructive pulmonary disease, and Familial amyotrophic lateral sclerosis-ALS. Detailed references can be found in James C. Sacchettini and Jeffery W. Kelly: Nature Reviews, Drug Discovery, Vol. 1 April 2002, 267-275.
In one embodiment, the subject is a human subject. Alternatively, the subject can be a mammal expressing an ApoE-CTD-like antigen with which a polypeptide of the invention cross-reacts. A polypeptide of the invention can be administered to a human subject for therapeutic purposes (discussed further below). Moreover, an anti-ApoE-CTD polypeptide can be administered to a non-human mammal expressing the ApoE-CTD-like antigen to which the polypeptide binds (e.g. a primate, pig or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of the polypeptide (e.g. testing of dosages and time courses of administration).
For in vivo embodiments, the contacting step is effected in a subject and includes administering the anti-ApoE-CTD polypeptide to the subject under conditions effective to permit both binding of the ligand to the plaque and the treating, e.g. the destruction of the plaque.
Methods of administering anti-ApoE-CTD polypeptides are described in “Pharmaceutical Compositions”. Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used.
The anti-ApoE-CTD ligands can be used directly in vivo to eliminate ApoE-CTD-containing plaques via natural complement-dependent cytoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). The polypeptides of the invention can include complement binding effector domain, such as the Fc portions from IgG1, −2, or −3 or corresponding portions of IgM which bind complement. The treatment can be supplemented by the addition of complement or serum containing complement. Further, phagocytosis of plaques coated with a polypeptide of the invention can be improved by binding of complement proteins.
Antibody-targeted amyloid plaques can be internalised by microglia through type A scavenger receptors (Melanie I. Brazil, Haeyong Chung, and Frederick R. Maxfield. Effects of Incorporation of Immunoglobulin G and Complement Component C1q on Uptake and Degradation of Alzheimer's Disease Amyloid Fibrils by Microglia J. Biol. Chem., May 2000; 275: 16941-16947). Alternatively, other mechanisms independent of the microglial Fc receptor might play a role in clearing diffuse, 3D6-immunoreactive, Thio-S-negative plaques and soluble Aβ moieties (Wilcock D M, DiCarlo G, Henderson D, Jackson J, Clarke K, Ugen K E, Gordon M N, Morgan D: Intracranially administered anti-Abeta antibodies reduce beta-amyloid deposition by mechanisms both independent of and associated with microglial activation. J Neurosci 2003, 23:3745-3751). Consistent with this hypothesis, Fc-knockout mice also showed reduction of plaque burden after Aβ immunotherapy (Das P, Howard V, Loosbrock N, Dickson D, Murphy M P, Golde T E: Amyloid-beta immunization effectively reduces amyloid deposition in FcRgamma−/−knock-out mice. J Neurosci 2003, 23:8532-8).
Also encompassed by the present invention is a method of killing or ablating which involves using the anti-ApoE-CTD ligand for prophylaxis. For example, these materials can be used to prevent or delay development or progression Alzheimer's disease, systemic amyloidosis or other amyloid disorders.
Use of the therapeutic methods of the present invention to treat Alzheimer's disease or systemic amyloidosis has a number of benefits. Since the polypeptides specifically recognise ApoE-CTD, other tissue is spared and high levels of the agent are delivered directly to the site where therapy is required. Treatment in accordance with the present invention can be effectively monitored with clinical parameters. Alternatively, these parameters can be used to indicate when such treatment should be employed.
F. Pharmaceutical Compositions
In another aspect, the present invention provides compositions, e.g. pharmaceutically acceptable compositions, which include an antibody of the invention formulated together with a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutical compositions” encompasses labelled ligands for in vivo imaging as well as therapeutic compositions.
As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvents, dispersion media, coatings, and the like. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion). Depending on the route of administration, the active compound, i.e. polypeptide may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-sold and solid dosage forms, such as liquid solutions (e.g. injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for administration of humans with antibodies. The preferred mode of administration is parental (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the anti-ApoE-CTD polypeptide is administered by intravenous infusion or injection. In another preferred embodiment, the anti-ApoE-CTD ligand is administered by intramuscular or subcutaneous injection.
The phrases “parenteral administration” and administered parentally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnod, intraspinal, epidural and intrasternal injection and infusion.
Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage.
The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e. the polypeptide) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
The antibodies of the present invention can be administered by a variety of methods known in the art, although for many applications, the preferred route/mode of administration is intravenous injection or infusion. For example, for therapeutic applications, the antibody can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5 or 1 mg/min to reach a dose of about 1 to 100 mg/m2 such as 7 to 25 mg/m2. The route and/or mode of administration will vary depending upon the desired results.
Pharmaceutical compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a pharmaceutical composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. No. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824 or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Of course, many other such implants, delivery systems and modules are also known.
In certain embodiments, the compounds of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB, they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g. U.S. Pat. Nos. 4,522,811, 5,374,548 and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhancing targeted drug delivery (see, e.g. V. V. Ranade (1989) J. Clin. Pharmacol. 29:685).
Dosage regimens are adjusted to provide the optimum desired response (e.g. a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parental compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody of the invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg. The antibody can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5 or 1 mg/min to reach a dose of about 1 to 100 mg/m2 or about 5 to 30 mg/m2. For antibody fragments which have lower molecular weights than an IgG, appropriate amounts can be proportionally less. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
The pharmaceutical compositions of the invention may include a “therapeutically effective amount” or a “prophylactically effective amount” of an antibody of the invention. The desired therapeutic result is typically a lessening or amelioration of one or more symptom of the disease or disorder from which the individual being treated is suffering. A therapeutic amount of an antibody of the invention may be an amount which serves to slow down or stop production of amyloid deposits, eliminate existing amyloid deposits, alleviate underlying disorders (that give rise to secondary amyloidosis), and relieve symptoms caused by heart or kidney damage. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of the polypeptide ligand to elicit a desired response in the individual. A therapeutically effective amount is also one is which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects. A “therapeutically effective dosage” preferably inhibits a measurable parameter, e.g. plaque formation or growth rate by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in humans. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit such inhibition in vitro by assays known to the skilled practitioner.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. The desired prophylatic result is the inhibition or delay in the outset or progression of symptoms associated with the disease it is intended to prevent in the individual being treated. Typically, since a prophylactic dose is used in subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
Also within the scope of the invention are kits comprising an antibody of the invention and instructions for use, e.g. treatment, prophylactic or diagnostic use. In one embodiment, the instructions for diagnostic applications include the use of the antibody to detect the form ApoE-CTD associated with plaques, in vitro, e.g. in a sample, e.g. a biopsy or cells from a patient having Alzheimer's disease or systemic amyloidosis, or in vivo. In another embodiment, the instructions for therapeutic applications include suggested dosages and/or modes of administration in a patient with Alzheimer's disease or systemic amyloidosis. The kit can further contain at least one additional reagent, such as diagnostic or therapeutic agent.
The following invention is further illustrated by the following Examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
EXAMPLES
Example 1
Antibody Library Composition
Antibodies that bind to ApoE found in plaques of amyloid disorders that do not bind to VLDL were selected from human phage antibodies in the Dyax phagemid library Fab300. Antibody diversity is present in the library used for the selections on CTD, the diversity in the light and heavy chains are composed as follows:
Heavy Chains
The heavy chain consists of one heavy chain gene segment (V3-23, or DP-47), in which diversity is created using synthetic oligonucleotides in certain positions in the HCDR1 and HCDR2. The distribution pattern is based on a diversity analysis of natural sequences. The appended HCDR3 diversity is derived from natural occurring sequences of the IgM-pool of B-cells from a series of autoimmune donors.
Light Chains
The light chain repertoire is derived from a pool of naturally rearranged light chains sequences, from the same source as the H-CDR3 diversity. This means that we can expect Vkappa and Vlambda genes, based on a diverse range of germine segments and with somatic mutations in or outside the CDRs.
Control Antibodies
The following anti-ApoE antibodies were used as controls:
3D12, a mouse antibody that binds to CTD, VLDL, LDL, ApoE2, ApoE3 and ApoE4 (Colabek et al. Biophysical J., 79:1008-1015);
E19, a goat antibody directed to CTD (Weisgraber (1986), J. Biol. Chem., 261: 2068-2076); and
6C5, a mouse antibody directed to NTD (Castano 1995) J. Biol. Chem., 270: 17610-17615.
Example 2
Preparation and Pretesting of Fibrils
Fibrils were extracted from spleen and kidney. Insoluble amyloid fibrils were extracted from human tissues by repeated rounds of mechanical homogenisation in cold 0.15M NaCl, 0.1% NaN3, with subsequent centrifugation in order to rescue the amyloid in the pellet. Finally the amyloid was dissolved/suspended in water and stored. Amyloid content was verified by Congo red staining of suspension smears. This method of extracting fibrils is known in the art, see for example Skinner et al. Prep. Biochem. 1982;12(5): 461-476. It was determined that bound and non-bound phage could be separated by washing with 5 Marvel-PBS-Tween (0.1%) washes, 2 PBS-Tween washes and 1 PBS wash.
Example 3
Preparation and Pretesting of Biotinylated CTD (bCTD)
CTD having the amino acid sequence shown in SEQ ID NO: 1 was used. Biotinylation was performed with sulfo-NHS-SS-biotin according to the method described by Pierce using molar ratios of CTD/biotin of 1/2 and 1/10. SDS PAGE revealed that 100% of the material was labelled. In mass-spectrometry at a ratio of 1/10, three to five of the five possible biotinylation sites were labelled with biotin. At a ratio of 1/2 bCTD showed one or two biotins per molecule which is favourable for keeping the structure of the molecule. All CTD and NTD used were labelled with the same protocol at a ratio 1/2 and tested in SDS-page.
Coated bCTD was prepared by coating BSA with biotin, washing, adding streptavidin and, after washing again, adding b-CTD.
To produce denatured bCTD, bCTD was treated with urea and, after washing, bound to streptavidin on biotinylated BSA. To be sure that the bCTD binds and stays on the beads during urea treatment, we measured the amount of bCTD before and after urea treatment and binding to the beads. No loss of bCTD was seen.
Example 4
Selections on Fibrils and ur-bCTD
In the selection strategy the aim was to select for antibodies binding to the complex form of CTD in amnyloid fibrils. A first round of selection was performed on fibrils from kidney, a second round of selection was performed on fibrils from spleen and rounds three and four of selection were performed on ur-bCTD. No enrichment was found in rounds two and three but in round four there was an enrichment of 1379. In a second strategy, three rounds of selections on bCTD were performed.
A pre-screening was then carried out to decide which round of selection to chose for a high throughput screen and which antigen should be used. There was no significant difference seen between screening on bCTD and ur-bCTD. It was, therefore, decided to carry out a high throughput screen on the ur-bCTD selection with streptavidin coated bCTD. As a negative control bBSA, streptavidin coated plates were used.
Example 5
Sequence and Binding Analysis
Screening of more than 2000 clones was performed on coated bCTD. ELISA was performed in an automated system. The same method was used to screen for NTD binders. Clones, binding to bCTD and not binding to bNTD were analysed further.
Sequencing was carried out for heavy and light chains genes for clones positive in the high throughput bCTD phage ELISA. Dyax's proprietary ‘Webphage’ software was used to analyse the sequences of clones.
752 clones were screened after round 3 (23 positives) and 216 clones after round 4 (216 positives) of the selections on fibrils and ur-bCTD. 940 clones were screened after round 2 of selections on bCTD. The number of positive clones in ELISA using bCTD was 463. The number of correct sequences obtained was 163.
To analyse the sequence data, comparisons of sequences at different levels were carried out, including the following:
(1) overall diversity=number of different sequences, will identify a clone as different if one amino acid difference is found (dubbed VH+VL diversity)=163
(2) heavy chain diversity=number of clones with a different heavy chain, ignoring the light chain sequence=152
(3) HCDR3 diversity=the number of clones with a different heavy chain CDR3=54 (+2 clones with amber HCDR3).
This three-level analysis was carried out for two reasons:
Firstly, it is generally accepted that the heavy chain, and, in that domain, its HCDR3 region, are of major importance for the epitope recognition of the antibody. All antibodies with an identical H-CDR3 sequence were grouped.
Secondly, the design of the diversity of the library is such that antibody variants that have an identical HCDR3 with mutations in the other CDRs of the heavy chain, and sometimes identical, sometimes different light chains are expected.
Example 6
Binding of Antibodies to bCTD and VLDL
To monitor binding to VLDL, VLDL was coated on a microtiter plate, and incubated with the test antibodies. A secondary antibody-HRP detection method is used to detect bound antibodies. Staining is performed with tetramethylbenzidine (TMB) and H2O2. Only non-bound antibody is washed away. Binding to VLDL will give a high signal in ELISA.
As a positive control we used monoclonal antibodies against ApoE. Two antibodies (3D12, E19), binding to CTD and VLDL, were positive in bCTD ELISA (FIG. 1A) and in VLDL ELISA (FIG. 1B). Another antibody (6C5) that binds to NTD does not bind to bCTD but binds well to VLDL. This NTD site is not covered by VLDL and could give a measure of coating quality of ApoE itself. Since of this antibody signal is high, we can conclude that enough ApoE is coated to perform the VLDL ELISA like it is.
For phage antibodies (FIG. 2), we made a classification in 3 groups: antibodies that are always positive (more than 3 times of negative phage binding), antibodies that are sometimes positive, sometimes negative (sometimes 2 times negative phage binding, sometimes negative) and negative antibodies. For the doubtful antibodies the ELISA is possibly not sensitive enough, or the antibodies are just not binding or probably the affinity is not high enough to see a high signal or there could be a cross-reaction with VLDL (epitope partially on VLDL, partially on covered CTD) etc.
Example 7
VLDL Assay Development and Automation
The VLDL ELISA was performed for all 203 bCTD positive phage clones. 6 clones were found which were always positive (more than 3 times the background). Other clones produced a signal higher than 2 times the background (FIG. 3). These clones are not excluded from further testing at this stage. Only 6 clones were positive, with a high signal, to VLDL. Tests were carried out 3 times, and the same results were obtained. In parallel binding of antibodies to bNTD was tested. No antibodies bound to bNTD. Clones that bound to VLDL were not tested further.
Example 8
Recloning of phase to Fab, Specificity Tests
Because of the low amount of VLDL binders in coated VLDL ELISA and because of the variable results of the VLDL competition ELISA, we batch recloned in parallel all different 157 clones from Fab on phage into soluble Fab. It was expected that many Fabs will not bind to bCTD because of their monovalent nature versus the multivalent phage, thus enabling low affinity binders to be excluded by means of Fab ELISA signals on bCTD.
After recloning, 85 antibodies bound specifically to bCTD. No new VLDL or NTD binders were found. The amino acid sequences of CDR regions of the VH and VL chains of these antibodies are shown in Tables 9 and 10.
Example 9
Epitope Mapping
Binding to identical epitopes is tested by monitoring competition between Fab and phage antibodies. A limited amount of phage and a maximal amount of Fab is added to an ELISA well coated with bCTD. After binding steps, phage is detected by a peroxidase reaction after incubating with an anti-M13 HRP antibody. Because of the high concentration of Fab added, phage directed to the same epitope as the Fab will be competed off and the Fab signal will be decreased.
Antibodies from the same VH-CDR3 group recognise overlapping epitopes. This criterion was used to exclude clones for immunohistochemistry (IHC): Only clones with the highest/slowest off-rate were tested. All clones not belonging to a big VH-CDR3 group were tested in IHC.
Antibodies 807A-M0026-F05 and 807A-M0027-E11 did not cross-react with each other. However, both cross-reacted with antibody 807A-M0028-A07, indicating that both antibodies recognise a similar but not the same epitope.
Antibody 807A-M0028-B02 possibly recognises another epitope than antibodies 807A-M0026-F05 and antibody 807A-M0027-E11.
Example 10
Off-Rate Measurements
To optimise Biacore measurements, we used a Biacore chip coated with streptavidin to bind bCTD. First we analysed antibodies 3D12 and E19 for binding to the chip. We also recloned a Fab that we recovered from the pre-screening (not binding to VLDL, positive in Fab ELISA). Mab 3D12 did not bind in Biacore, probably due to low affinity. Ab E19 and non-purified Fab 1F7 (dialysed periplasmic fraction) did bind to the bCTD chip. As a control we used a channel coated with bBSA; neither antibody bound to this surface.
Off-rate ranking of selected Fabs was determined with this bCTD chip using periplasmic extracts (important for ranking clones, now and during affinity maturation studies). Table 1 shows a representative list of off-rates.
Affinity determination was determined on a low density of bCTD on the chip using purified Fab fragments.
Example 11
Immunohistochemistry
Immunohistochemistry (IHC) on the antibodies with the slowest off-rate of the identical VH-CDR3 groups and all antibodies having a different VH-CDR3 (single clones) were tested in IHC performed on frozen tissue slides.
For IHC and affinity measurement purified Fabs were used. Antibody fragments (Fabs) were expressed in bacteria (typically in 400-ml cultures, 4 hours IPTG induction) and purified from periplasmic extracts by Immobilized Metal Affinity Chromatography (IMAC). Periplasmic extracts were prepared by “osmotic shock” treatment of the bacteria. The samples were loaded on a 1 ml Co-IDA column and eluted with a 0-150 mM linear gradient of imidazole. Protein preparations were dialyzed against PBS and analyzed by non-reducing SDS-PAGE.
Only 3 antibodies bound as Fab on AD plaques: 807A-M0027-E11, 807A-M0028-B02 and 807A-M0026-F05.
Antibody 807A-M0027-E11 detects Alzheimer's Disease (AD) plaques in IHC. It is important that antibodies specifically recognise plaques in tissue and not to Apolipoproten E exposed in serum. Typically, an antibody of the invention binds to plaques of at least two patients having AD and to patients potentially also having systemic amyloidosis. Therefore, IHC was performed in the presence of fresh plasma. In IHC staining with 807A-M0027-Eli, as phage and as Fab, in the presence of even 50% plasma, the signal was not quenched. Also the addition of VLDL in solution did not change the staining. This is in contrast with the 6C5 control antibody, directed to NTD, the signal of which was quenched by fresh plasma or VLDL solution. SFab antibody 807A-M0028-B02 stained positive on AD plaques and also astrocytes. SFab antibody 807A-M0026-F05 stained AD plaques weakly. The staining pattern for this antibody is not very strong, caused by low affinity of this antibody. The antibodies were positive in tissues of more than 1 patient.
Other sFab antibodies: 807A-M0039-C10, 807A-M0037-D01, 807A-M0046-A06 and 807A-M0039-C10 only detected astrocytes on AD brain tissue.
Example 12
Affinity Measurement of sFabs Binding to Plagues in IHC
The three Fabs 807A-M0027-E11, 807A-M0028-B02 and 807A-M0026-F05 were extensively studied in Biacore analysis. First bCTD was coated on a streptavidin chip, then sFabs were run over the chip at different concentrations and binding resonance units (RU) were measured. As a negative control, one channel of the chip was saturated with biotin-BSA. FIG. 4A shows the analyses of sFab antibody 807A-M0027-E11 on bCTD resulting in an affinity is 47.8 nM. The affinity of sFab antibody 807A-M0028-B02 showed a similar pattern, with an affinity of 179 nM (FIG. 4B). Antibody 807A-M0026-F05 has such a low affinity (in the μM range) that it is difficult to measure (FIG. 4C).
In contrast, when sFabs were coupled to a CM5 chip via an anti-Fc antibody, no binding was observed for antibody 807A-M0027-E11 (see FIG. 4D), nor for antibody 807A-M0028-B02.
The different results reversing the coating and analyte, in the two different Biacore measurements suggest that both, antibody 807A-M0027-E11 and 807A-M0028-B02 only bind to coated bCTD and not to bCTD in solution.
Example 13
Antibody Reformatting Expression and Purification of IgGs
Antibody (Batch) Reformatting to IgG1
85 clones showed specific binding to CTD as soluble Fab. Of these the 30 candidates that had been chosen for IHC studies were reformatted to complete human IgG1 antibodies.
A pool of 157 CTD-specific Fabs, that contained all 85 “soluble Fab binders”, was used for simultaneous, restriction digestion based batch-reformatting into the human IgG1 expression vector pBh1.
The batch-reformatting strategy involved two cloning steps, and is illustrated in FIG. 5. In the first step, complete Fab fragments are inserted into pBh1. In the second step, internal/regulatory sequences are exchanged.
To “re-identify” the initial Fabs, about 300 individual clones were analysed by DNA-sequencing. 72 of the 85 “soluble Fab binders” were found back. Notably, 29 of 30 of the prioritised candidates chosen for IHC (as phage and soluble Fab) were obtained as IgG1 constructs by batch reformatting.
11 of the 13 remaining “soluble Fab binders” could be reformatted individually into the human IgG1 expression vector pRh1. Identity of the reformatted antibodies to their Fab counterparts was verified by sequencing. Besides initial PCR-amplification of the complete Fab-insert, the cloning approach is identical with the batch-reformiatting strategy depicted in FIG. 5.
Expression and Purification of IgG1 Antibodies
Reformatted IgG1-antibodies were expressed in transiently transfected HEK293T cells. Antibodies were purified from culture supernatant of  ̃5×106 transfected cells (per flask), kept in culture for about one week. Purification was carried out by Protein-A-based affinity chromatography. Purified antibodies were dialyzed against PBS and analysed on SDS-gel under reducing and non-reducing conditions.
Biotinylation of IgG Antibodies
Biotinylation of antibodies was performed in PBS, incubating the purified antibodies for 2 hours with a 15-fold molar excess of Sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. The level of biotin-incorporation (i.e. the average number of biotin groups per antibody molecule) was determined using the HABA [2-(4′-hydroxyazobenzene) benzoic acid] method (Pierce). Using this approach, we found that all biotinylated antibodies contained 3 to 4 biotin groups per molecule.
Example 14
Specificity Tests with IgG1 Antibodies
Binding of IgG1 Antibodies to Other Species
Cross-reactivity of the human antibodies on bCTD of these species was tested to try to identify antibodies that can be studied in mouse and primate models.
The antibodies (807A-M0028-B02, 807A-M0027-E11, 807A-M0026-F05) that bound to plaques in AD tissues as sFab, were reformatted to hIgG1 and tested for their binding capacity on recombinant mouse CTD (mbCTD), recombinant primate CTD (pbCTD) and recombinant human CTD (hbCTD) that was biotinylated.
Antibodies 807A-M0028-B02 and 807A-M0027-E11 (FIGS. 6A and 6C) did bind to CTD of the three different species. Both antibodies bound to the same extent suggesting that the epitopes recognised by the antibodies are the same in these species.
Antibody 807A-M0026-F05 (FIG. 6B) binds to pCTD and hCTD but not to mouse CTD, suggesting that the antibody is directed against an epitope that is not present in mice or that concentration used is not high enough. The low O.D. in this ELISA can be explained by the low affinity of this antibody.
Binding of IgG1 Antibodies to bNTD
807A-M0027-E11 and 807A-M0026-F05 did not bind to bNTD. Antibody 807A-M0028-B02 did bind at very high concentrations (10 and 5 μg/ml). To check if this was related to specific binding, we tried to compete with 1000 times more bNTD in solution (540 ug/ml in solution, 0.5 ug/ml coated) (FIG. 7). The non-specific signal did not decrease in competition. The signal is most probably due to the high amounts of antibody added, as also seen for the anti-MUC1 antibody PH1 (FIG. 7).
Binding of IgG1 Antibodies to VLDL
Binding of antibodies to coated VLDL was tested in ELISA. Unlike the phage and the Fabs of the plaque binders, antibodies 807A-M0028-B02 (FIG. 8) and 807A-M0027-E11 are binding to VLDL to the same extent. When compared to the binding of a non-CTD binder (PH 1, which is a MUC 1 binder) this binding seems to be specific.
Because VLDL is composed of ApoE and a lipid, the coated VLDL could change its conformation during treatment in ELISA and it could be that the CTD is not covered by the lipid anymore. Therefore we did a competition test with the antibodies in solution, bCTD bound to the plate and an excessive amount of CTD or VLDL in solution (FIG. 9). In this assay we could not see inhibition with VLDL for the human antibodies (FIGS. 9A and B) and little inhibition with CTD for antibody 807A-M0028-B02 (FIG. 9B). In contrast, the commercial antibodies 3D12 and E19 directed against CTD were clearly inhibited by VLDL as well as by CTD. The 6C5 monoclonal directed to NTD did not bind in this assay.
These results suggest that the human antibodies (807A-M0028-B02, 807A-M0027-E11) are not binding to VLDL in solution and recognise CTD in solution to a lesser extent than coated bCTD.
Conclusion
Two of the three antibodies binding to plaques in AD (807A-M0028-B02, 807A-M0027-E11) are cross-reactive with pCTD and mCTD. The third antibody (807A-M0026-F05) does not cross-react with mCTD and binds pCTD. The antibodies of interest do not bind to NTD. Two antibodies (807A-M0028-B02, 807A-M0027-E11) bind to coated VLDL, when high antibody concentrations were used. Antibody 807A-M0026-F05 does not show these properties but this could be related to the affinity of the antibody.
Example 15
Biacore Analysis
To compare Fab and IgG1 binding and to study the nature of binding of the human antibodies on CTD in solution, Biacore was extensively used.
Comparison of Fabs and IgGs in Biacore
FIGS. 10, 11, 12 and 13 summarize the results of the Biacore analysis of the CTD-specific clones 807A-M0026-F05, 807A-M0027-E11 and 807A-M0028-B02. As expected all three plaque binders bind better to bCTD on the chip as IgG1 compared with their original Fab format. IgG1 Antibody 807A-M0027-E11 binds 3 times better than antibody 807A-M0028-B02 while antibody 807A-M0026-F05 binds with a very low micro molar avidity to the chip.
The avidity measured (Table 2) is higher when measured 50 seconds after the injection of the antibody is stopped as compared with the avidity measured immediately after injection stop. This difference is probably due to the rebinding of the antibody to the chip when free bCTD is available on the chip.
Binding of Antibodies to Captured CTD
Because of the inconsistency of VLDL ELISA in which the human IgG1 antibodies bind to coated bCTD/VLDL but not CTD/VLDL in solution, additional Biacore experiments were performed (see also Biacore on Fabs). In these experiments bCTD or ApoE was captured. First anti-hFc antibody was coupled to the chip, followed by binding of the specific antibodies, followed by the injection of bCTD or ApoE.
FIG. 12 shows that Fab antibody 807A-M0027-E11, indirectly coupled to the chip, does not bind bCTD (280 nM). The same curves (data not shown) were obtained by using Fab antibody 807A-M0028-B02 and when IgG1 of both antibodies was used. The test was performed with ApoE originating from human serum. Only a very small amount of ApoE (17 RU, <1%) was bound to the antibody (FIG. 13).
Conclusion
In Biacore, IgG1 antibodies 807A-M0027-E11 and 807A-M0028-B02 bind to coated bCTD with nM avidity while IgG1 antibody 807A-M0026-F05 binds with μM avidity. Affinity measurements on antigen in solution do show that both antibodies 807A-M0027-E11 and 807A-M0028-B02 do not capture bCTD nor ApoE efficiently. These results confirm the results seen by VLDL/CTD competition ELISA: antibodies 807A-M0027-E11 and 807A-M0028-B02 bind better to coated bCTD than to CTD in solution.
Example 16
Additional Testing to Study Binding of Antibodies to Natural CTD to Peptides
SDS-PAGE Analysis of the Purified hApoE
The purified, hApoE was analyzed by reducing SDS-PAGE followed by Coomassie staining. As expected, the protein migrated as one major band of  ̃35 kDa, but there was also a broad band at  ̃70 kDa and a faint smear from 70 kDa to  ̃200 kDa. Both the 35 kDa band and the higher molecular weight species were shown by western-blot to contain hApoE.
Immunoprecipitation of Purified hApoE
Purified hApoE was immunoprecipitated with 807A-M0028-B02, 807A-M0027-E11 and 807A-M0026-F05. As a positive control, we used E19, a goat anti-hApoE antibody. As a negative control, we used the PH1 antibody. Untreated, purified hApoE was also included as a reference. The samples were analyzed by SDS-gel, the proteins were transferred to a nitrocellulose membrane and hApoE was detected by western-blot.
As expected, the E19, the 807A-M0028-B02 and the 807A-M0027-E11 antibodies were able to specifically immunoprecipitate hApoE, although not very efficiently. Interestingly, the E19 antibody seemed to be specific for the 35 kDa band, whereas the 807A-M0028-B02 and 807A-M0027-E11 antibodies were more specific for the high molecular weight species. The 807A-M0026-F05 was by contrast unable to immunoprecipitate hApoE, probably due to the low affinity of the antibody.
Immunoprecipitation of Cell Lysates
Cell lysates of PBMC were immunoprecipitated using 807A-M0028-B02, 807A-M0027-E11 and 807A-M0026-F05, as well as E19 (positive control) and M43G5, M43F8, PH1, A2, herceptin and a human IgG1 Kappa Myeloma antibody (negative controls). Samples were analyzed by SDS-PAGE under reducing conditions followed either by silver staining or Western-blot.
Only E19 immunoprecipitated a band of the expected size (i.e. 35 kDa). The material immunoprecipitated with E19 also gave a very faint signal in Western-blot. This suggests that some hApoE is captured by E19 in the cell lysates, although this finding must be regarded with care since (1) 807A-M0028-B02 and 807A-M0027-E11 did not immunoprecipitate any hApoE from the cell lysates, (2) 807A-M0026-F05, which was shown to be unable immunoprecipitate hApoE, also gave a weak signal in Western-blot and (3) A2 and 807A-M0043-F08, two irrelevant antibodies, gave strong signals in Western-blot.
Importantly, the three antibodies investigated here (807A-M0028-B02, 807A-M0027-E11 and 807A-M0026-F05) did not seem to immunoprecipitate any major component of the cell lysates. The background for 807A-M0027-E11 was a little bit higher, but not higher than e.g. PH1. These results indicate that the overall specificity of the antibodies, 807A-M0028-B02, 807A-M0026-F05 and 807A-M0027-E11, is due to the binding to CTD.
Immunoprecipitations of VLDL
Binding of antibodies to VLDL was also tested by immunoprecipitation. 10% VLDL was used in these tests. As detection antibody 6C5 was used. After 5 minutes development of a Western blot with the ECL method, no ApoE was detected for the human antibodies of interest. After overnight development antibody 807A-M0027-E11 and 807A-M0028-B02 were detected as faint bands when compared with the VLDL control (non-immunoprecipitated VLDL, 10% of the amount that was used for immunoprecipitation). Immunoprecipitation with the 6C5 antibody showed a more extensive band than with the human antibodies.
Discussion
807A-M0028-B02 and 807A-M0027-E11, but not 807A-M0026-F05, preferentially immunoprecipitate a high molecular weight from of hApoE purified from plasma. The nature of these species remains unclear, although it is clear that they contain hApoE and must form very stable complexes. These antibodies do not interact significantly with major cellular components. Immunoprecipitation of VLDL with the antibodies is possible, although the amount might be very low.
Example 17
In Vivo Studies
Mice were bred to express in the brain the human gene for amyloid precursor protein (APP): Swedish mutation K670N,M671L, APP Line 2576, driven by the hamster prion promotor (Hsia et al Science 1996, 274:99-102), alone or in combination with a mutated human presenilin 1 (PS1):M146L driven by the platelet derived growth factor (Duff et al, Nature 1996, 383 (6602):710-3, Holcomb et al, Nature Med 1998, 4:97-100).
For studies requiring expression of human ApoE mice were bred that either expressed the human ApoE4 driven by the glial fibrillary acidic protein (GFAP) promotor with or without the mouse ApoE gene knocked out (Sun et al, J Neurosci, 1998, 18:3261-3272) in combination with humAPP:Swe and humPS1:M146L or humAPP:Swe only.
Monoclonal antibodies (mAb) of the human immunoglobulin G1 (hIgG1) isotype to C-terminal domain (CTD) of Apolipoprotein E (ApoE) were injected intra-peritoneally at a concentration of e.g. 10 mg/kg in non transgenic or transgenic mice. Some mice were injected once and sacrificed 2 days after injection and some were injected twice, with the repeated dose injected after 2 days, and then sacrificed after an additional 2 days i.e. 4 days after the initial injection. The concentration of injected antibody was monitored by ELISA to CTD binding hIgG. Brains from mice injected with a streptavidin specific monoclonal antibody served as negative controls. The sampled brains were immediately frozen to −70° C. and then subjected to freeze sectioning.
Staining for presence of human IgG in brain sections showed homogeneously stained plaques evenly spread trough cortex and hipocampus. No other brain structures showed staining after in vivo exposure. In contrast, several structures along with the amyloid plaques were stained after ex vivo exposure. 70% of the plaques that were accessible for staining ex vivo by anti-ApoE CTD antibody or with a mAb to Ab were stained after the 2-day (n=3) or 4-day (n=3) in vivo exposure with the anti-ApoE CTD antibody. The in vivo exposure for 2 or 4 days at the given dose did not saturate the available binding sites as indicated by the additional staining intensity obtained after ex vivo addition of more anti-CTD antibody.
Antibodies, as macromolecules in general, do not pass freely over the blood brain barrier (BBB). The passage of IgG is considered to be very limited and concentrations in CSF under 0.5% of the plasma concentration has been reported (Elovaara et al, 1987 Eur Neurol 26:229-34, Ganrot & Laurell 1974, Clinical Chemistry 20:571-3). Staining for presence of the intraperitoneally injected human IgG in brain sections revealed that the ApoE CTD specific antibody reached the cerebral plaques evenly throughout the different brain regions in these transgenic mice indicating sufficient BBB passage for staining by immunohistochemistry (IHC) technique. Alzheimer's disease (AD) plaques are complex structures varying in size and density. The cerebral amyloid plaques found in these transgenic mice are considered to represent the small diffuse and medium size plaques found in AD. The plaques were homogeneously stained indicating that the mAb did not only reach the outer layer of the plaques but penetrated the whole plaque structure. 70% of the plaques accessible for staining ex vivo were stained after the in vivo exposure. Considering that the dose of 10 mg/kg administered interperitoneally did not saturate the available plaque-binding sites, a non-saturated level of antibodies may still result in an antibody mediated plaque breakdown by FcR bearing phagocytic cells.
Example 18
Reformatting of Antibodies to Mouse IgG2a
For in vivo testing the variable regions of antibodies 807A-M0028-B02, 807A-M0027-E11, 807A-M0026-F05 and a control antibody (anti-Streptavidin clone A2) were recloned into vector that contains mouse IgG2a constant regions of the heavy chain and mouse Ckappa and the variable regions of antibodies.
These clones were transferred from a human IgG1 expression vector (pBh1) to a construct for expression of mouse IgG2a antibodies (pRmk2a), that besides the constant heavy chain region, also contains the mouse constant kappa light chain gene. The VL and VH regions were lifted from the human IgG1 expression plasmid via PCR, and cloned sequentially into pRmk2a. VL was inserted as an ApaL1/BsiW1 fragment, 3′ of the antibody leader and 5′ of the constant kappa gene. In case of the VH region, the 5′ adjacent IRES motif was also included in the PCR amplification product; an Asc1/Nhe1 fragment was inserted in pRmk2a. Integrity of the constructs was verified by DNA sequencing. The cloning strategy is depicted in FIG. 14.
Example 19
Preparation of Peptides
10 peptides (length 16 amino acids) covering the full ApoE CTD were synthesized.
The peptides contain an 8 amino acid overlap between each other as shown in FIG. 15. The peptides contain an S—S Biotin group that enables binding to Strepatavidin (magnetic beads selections). In addition each peptide contains a Cystein that can be coupled to a carrier protein (BSA).
Peptides were solubilised in dimethylformamide (DMF), and subsequently diluted in water. All peptides, except peptide 4 were soluble in DMF at a concentration of less then 10%. Coupling was performed in 10% DMF for all peptides, except for peptide 4 which was coupled in 30% DMF. An excess of maleimide-activated BSA was used to bind to the peptides. After incubation an excess of Cystein was used to occupy possible free cysteins. The BSA coupled peptides (bpeptide-BSA) were used for selections.
Example 20
Binding of Antibodies to Overlapping Peptides
Antibodies 807A-M0026-F05, 807A-M0028-B02 and 807A-M0027-E11 identified in Example 5 were tested for their binding to overlapping peptides. This was done firstly to test which peptides could be preferentially used in further selections: bpeptide-BSA or bpeptide and secondly to see whether the antibodies selected in Example 5 were binding to the overlapping peptides and if so, whether the epitopes they recognised were different and supported the epitope mapping by competition results as performed in Example 9. Both human antibodies and mouse antibodies were compared in this way.
For the human antibodies, peptide mapping was performed with IgG1 as well as with the phage displayed Fab. For example, clone 807A-M0026-F05 recognised bpeptide-BSA 4 and 8 as phage Fab fragment as well as whole IgG1. The ELISA was not sensitive enough to show binding to bpeptide 4 and 8 for this antibody. Therefore, we decided that it would be best to start the selections on bpeptide-BSA to capture the majority of peptide binders and then, if necessary, use the bpeptide in a later round of selection. Remarkably, antibody 807A-M0026-F05 bound better to CTD than to peptide compared with the murine monoclonal antibodies. Antibody 807A-M0028-B02 bound to peptide 4. Antibody 807A-M0027-E11 did not significantly bind to any of the overlapping peptides.
In Example 9 we found that the epitope recognized by antibody 807A-M0026-F05 and antibody 807A-M0027-E11 was covered by a large group of antibodies. Both antibodies did not compete with each other. Since the affinity for CTD for antibody 807A-M0026-F05 is very low as compared with antibody 807A-M0027-E11, one would expect that, if they recognise the same epitope, antibody 807A-M0027-E11 would have bound more strongly to peptides 4 and 8 than antibody 807A-M0026-F05. Therefore, one could conclude that both antibodies recognise related but not identical epitopes. Antibody 807A-M0028-B02 bound to peptide 4 and was different from the antibody group of the two other antibodies in competition epitope mapping and could recognise a different epitope.
We also tested control mouse antibodies on overlapping peptides. Antibodies 3H1, 12D10 and E19 bind to peptides 3, 10 and 5+10 respectively. In contrast with the human antibodies, all control antibodies bind to about the same extent to peptide and bCTD.
Example 21
Selections and Screening on Peptides
Three rounds of selection were carried out on 10 overlapping biotinylated peptides conjugated to BSA (b-peptide-BSA) and one round of selection on the corresponding biotinylated peptides (b-peptide). Selection was performed on 10 individual peptides using Streptavidin-magnetic beads. To handle this high number of selections in between selection rounds no titration of input/output was performed (liquid amplification).
The procedure used is set out in FIG. 16. First, three rounds of selection for binding to bpeptide-BSA was carried out using the automated Kingfisher system. Pre-screening of round 2 and 3 with Fab-displayed on phage showed that the frequency of positive clones was low in round 2 and that many clones were binding to BSA in round 3, despite extensive depletion and subtraction on BSA. Most likely the binders were directed to the linker molecules on the BSA that we used for coupling the peptides. Therefore another round 3 selection on bpeptides was performed. For this selection background binding was negligible.
To reduce sequencing efforts, phage-Fab clones were batch reformatted to produce sFabs and the large screening of the clones (ELISA and sequencing) was performed at sFab level. 307 antibodies were found positive in ELISA of which 46 were unique as was determined by sequencing as shown in Tables 11 and 12.
Example 22
Selections and Screening on Fibrils and Peptides
Two rounds of selection were carried out on fibrils originating from an organ with amyloid plaques, 2 rounds on 10 corresponding b-BSA-peptides and 1 round on 10 corresponding b-peptides.
The procedure used is summarised in FIG. 17. Round 3 and round 4 were performed on individual bpeptides-BSA. After pre-screening few positive clones were found. Therefore a fifth round of selection was carried out using bpeptides. In a pre-screen, positive clones for peptide 4 and 8 were found. For these two selections we screened sFab after batch reformatting. In total 390 sFabs were screened, 109 were positive in ELISA and 4 clones were unique. The amino acid sequences of the VH and VL chains of these unique clones are shown in Tables 11 and 12. In these strategies most clones did show unique VH-CDR3s (Table 12), and high enrichment was found: one clone was enriched 148 times and was found in strategy B1 as well as in strategy B2. This is in contrast with selections of Example 5.
Few clones (Table 13) bound to bCTD, which makes them unique as compared to results in Example 20 where bCTD binders bound weakly or not at all to peptides. In the selection campaigns of Examples 21 and 22 no Fabs were binding to VLDL nor to NTD.
11 Fabs were found positive in IHC, 10 of these Fabs originate from the selection campaign of Example 11 and one originates from the selection campaigns of both Examples 21 and 22.
Example 23
Selections and Screening on ur-bCTD and Peptides
Two rounds of selection were carried out on urea treated biotinylated CTD (ur-bTD) followed by two rounds on fibrils originating from an organ with amyloid plaques. The procedure used is summarised in FIG. 18.
Two rounds of selection on urea-CTD antigen were performed followed by 2 selection rounds on fibrils 1 from an AD patient. In this strategy pre-screening was done after the 3rd and 4th round of selection. Frequencies were 82/95 and 83/95 respectively. During pre-screening we used ur-bCTD and bCTD and no difference was found between both types of antigen. After the third selection round, many clones were retained that bound to bCTD but not to fibrils while after 2 rounds of selection on the fibrils (4th round of selection) the chance that these binders are retained was less. Therefore, large scale screening was performed on the 4th round of selection. 950 sFabs were screened on ur-bCTD. 233 clones were positive in ELISA as soluble Fab, 83 were unique. The amino acid sequences of the VH and VL chains of these uniques Fabs are shown in Tables 14 and 15. In VLDL ELISA, 5 Fabs bound were positive or had questionable binding.
No Fabs bound to bNTD.
Many Fabs belonged to large families of identical VH-CDR3 groups; one individual antibody was enriched 247 times. Testing in IHC was performed on individual clones (not belonging to a large VH-CDR3 group) and clones belonging to a large VH-CDR3 group that were selected for a slow off-rate.
In a first IHC screen, 6 Fabs bound to plaques of AD patients. The properties of the bCTD binders are summarised in Table 17.
Example 24
Production of Candidate Clones as Soluble Fabs for IHC
Fabs were produced for further analysis. ELISAs were performed on periplasmic fractions of 100 μl cultures. Biacore off-rate measurement on periplasmic fractions of 50 ml cultures of all clones found in tables with Biacore results.
About 90 soluble Fab proteins were produced for testing in IHC. At least 10 μg was required for initial analysis. Because of wide variation in soluble Fab expression levels, the protein had to be purified either from the peripheral extract of 50 ml bacterial cultures, or from peripheral extracts of 400 ml cultures, by IMAC chromatography, using 96-well filter plates and a vacuum manifold. The yields mainly ranged between 10-100 μg.
Large Scale Production of IHC Positive Soluble Fabs
15 of the candidate Fabs turned out to be positive/potentially positive in IHC on plaque tissue. Of these clones more soluble Fab protein were prepared for additional testing.
Soluble Fab proteins were prepared from the periplasmic extracts of 400 ml bacterial cultures.
Example 25
Epitope Mapping Comparison with Preliminary IHC Results
High throughput Fab screening was performed on the peptides they were selected on and also on a non-overlapping peptide as a control. In epitope mapping all peptide positive antibodies were screened for their binding reactivity to all other peptides. Table 13 contains detailed results.
Two Fabs bound to all peptides (most likely to the BSA-linker) and were considered as non-specific.
No Fabs were identified that were specific for peptide 5 and 10 (although the pre-screening Fab on phage showed some positive binders).
49 different Fabs bound specifically to peptides. 39 of the specific Fabs only bound to the peptide they were selected on. One Fab selected on peptide 1 also recognised peptide 6. 3 Fabs originating from the peptide 4 selection also bound to peptide 9 and 3 Fabs originating from the peptide 9 selections recognised peptide 4. Further, 3 peptides selected on peptide 8 also bound to peptide 4. Only 9 Fabs bound also to bCTD. This suggests that most of these Fabs bind to an epitope that is not present in the recombinant bCTD and recognise another (possibly stretched) structure that could potentially also be found in plaques. The region that covers peptide 3 and peptide 4 is recognised as a ‘selection dominant epitope’, containing 39 of all 49 specific Fabs and 8 of 9 bCTD binders. Interestingly, this area of ApoE is thought to be involved in the binding to VLDL particles.
Hypothesis Taking into Account for The IHC Screen
39 of the 49 specific Fabs bound to peptide but not to bCTD. This suggests that these Fabs are not likely to recognize natural Apo-E contained in for example VLDL particles. If such a Fab would bind in IHC it could indicate that such an epitope is unique and only found on plaques and that these Fabs would be important leads for further investigation.
Indeed, preliminary IHC data shows that four of these Fabs possibly bind to tissue in AD patients. These four Fabs all bind to peptide 4 and not to the overlapping peptides, suggesting that they recognise similar (overlapping) epitopes (group 1), probably epitope containing amino acids of LVEDMQRQ (SEQ ID NO: 12) or a secondary structure only present in peptide 4 and plaques.
Two other Fabs, positive in IHC and selected on peptide 4, bind to peptides 4 and 9 and to a conformation that is not present or not as prevalent in bCTD. Possibly the epitope for these two antibodies include sequence MQRQWAGL (SEQ ID NO: 13, group 2).
Another Fab possibly positive in IHC, selected on peptide 9, only recognises peptide 9 and not overlapping peptide nor bCTD and could recognise either the epitope WAGLVEKV, (SEQ ID NO: 14) or a conformation only present in peptide 9 (group 3) and plaques.
One Fab binds to peptide 1, 6 and bCTD. This epitope (RTRDRLDE, SEQ ID NO: 15) is not predicted to be inside of the binding site of VLDL (group 4).
Two antibodies, obtained from selections on peptides 4 and 9, recognise both peptides (epitope MQRQWAGL, SEQ ID NO: 13) and CTD and therefore are different from Fabs of group 2 (group 5).
One antibody, selected to peptide 8, binds to peptide 4, 8 (epitope WFEPLVED, SEQ ID NO: 16) and bCTD (group 6).
Thus, according to this hypothesis the Fabs identified in Examples 21 and 22, can be divided into six different groups of Fabs that each recognize distinct epitopes.
Example 26
Biacore Off-Rate Analysis of the Fabs Identified in the Strategies of Examples 21 and 22
Off-rate analysis of soluble Fabs was performed on all 10 peptides. Periplasmic fractions from all unique Fab of Examples 21 and 22 were made and tested. The results confirm the epitope mapping by ELISA.
Fabs that bind to more peptides and/or to bCTD most often show the about the same off-rate for those molecules. In contrast, RU's (measure for the amount of antibody bound) are often highest for the peptide to which the Fabs were selected on.
The strategies of Examples 21 and 22 did not result in the identification of antibodies belonging to large families of identical VH-CDR3s. Therefore, off-rate measurements were not used as a criterion for IHC.
Example 27
Epitope Mapping of the Antibodies of Example 23 and Comparison with Preliminary IHC Results
In the automated screening, Fabs were screened for their binding reaction towards ur-bCTD and streptavidin BSA as negative control. No antibodies bound to NTD. Table 16 contains detailed results.
In total 81 different antibodies, binding to ur-bCTD and CTD were found. 5 of those antibodies bound to coated VLDL. None bound to bNTD. 20 antibodies, also bound to peptide. As in the strategies of Examples 21 and 22 we found a selection dominant epitope around peptides 3 and 4.
Five antibodies of the 81 bound in IHC. Only one of these antibodies bound to peptide. This antibody binds with low RU (Biacore) to peptide 4 and high RU. Interestingly, this antibody was also found using the strategies of Examples 21 and 22. The other four antibodies could be compared with antibodies 807A-M0028-B02 and 807A-M0027-E11 of Example 5.
Example 28
Reformatting of Candidate Fabs to Human IgG1
Most of the IHC positive clones described above can be individually reformatted to the Dyax hIgG1 expression construct pBh1 in two restriction endonuclease based (“cut and paste”) cloning steps (see FIG. 5).
With the exception of the amber-stop containing clones, 807B-M0079-D10 (807B-M0027-D08) and 807B-M0081-A11 (807B-M0081-F12), and the clone 807B-M0009-C03, reformatting of Fab to IgG was carried out and the IgGs transiently expressed in Hek293T cells.
The amber-stop mutation in the CDR2 of 807B-M0079-D10 is corrected on the “phagemid level”, before the clone is reformatted using the procedure outlined above.
The amber-mutation at the 5′-end of VL of 807B-M0081-A11 is repaired using a different reformatting strategy/“PCR-based reformatting to the hIgG1 expression construct pRh1”. Due to the fact that the amber-stop mutation lies within the sequence of our “CJ-kappa-lifting primer”, the stop mutation is corrected during PCR amplification of the Fab fragment. The cloning strategy of PCR/Fab fragments to pRh1 is the same as the “cut and paste” approach to pBh1.
Example 29
Conclusions
229 candidate Fabs binding to CTD were isolated from a variety of selection procedures with Dyax' human Fab300 library. Two of the procedures included selections on peptide (Examples 21 and 22). In the selection procedure of Example 23 the successful selection of Example 5 was reversed by first selecting on ur-bCTD and then on fibrils. Also in contrast with Example 5, we did not screen phage but first performed a batch recloning from phage Fab to Fab.
Very few (five) were reactive, as Fab antibody, with coated VLDL. No Fab was positive for bNTD.
In the strategies of Example 21 and 22, some clones were enriched and we observed VH-CDR3 groups with few individual clones.
Fab antibodies from the strategies of Example 21 and 22 recognise a selection dominant epitope around peptides 3 and 4. In the strategy of Example 23, the same dominant epitope is found.
In IHC screening, 15 antibodies were found positive for binding to plaques in IHC in a first screen. An overview of the characteristics of the Fab clones positive in the first IHC screen is shown in Table 17.
Example 30
Properties of IgG1 of the 15 Fabs Positive in IHC
The 15 Fabs which were positive in Fab-IHC were reformatted to IgG1. Of these antibodies, nine were found positive in IgG1-IHC.
Almost all IgG antibodies bound to peptide in peptide epitope mapping. This is probably due to the higher avidity of the IgG1 as compared to the Fab. For the antibodies selected on peptide 4 which only bound to peptide 4 as Fab, some also bound to peptide 9 as IgG. For the antibodies originally from the screen described in Example 23, the IgG antibodies bound to peptides 4 and 8, peptide 9 or peptide 7 only one antibody, 807B-M0083E11, did not bind to any peptide.
Human CTD, mouse CTD and primate CTD were compared to each other. Several IgG antibodies bound to bCTD of each of the three species.
Although there was no binding to coated VLDL in Fab ELISA, in IgG1 ELISA several but not all antibodies bound to VLDL.
All IgG antibodies showed an improved binding compared to Fabs in Biacore analysis when bCTD was coated on the chip.
The IgG1 results are summarised in Table 18.
Example 31
Effect of VLDL on Binding of 807A-M0028-B02 to CTD in Amyloid Deposits
To analyse binding of 807A-M0028-B02 to CTD in amyloid deposits in presence of lipoprotein particles, immunohistochemistry was performed in presence or absence of VLDL to see whether the presence of VLDL would lead to a decreased staining intensity of plaques.
The 807A-M0028-B02 antibody was serially diluted and mixed with VLDL prior incubation of both APP/PS1 and human AD brain sections. No quenching of signal was observed even at the lowest antibody concentration (0.04 μg/ml). In contrast, a commercial antibody to CTD (3H1) was completely quenched already at a high concentration of antibody (5 μg/ml) (FIG. 20).
Example 32
Effect of 807A-M0028-B02 Antibodies on Phagoeytic Activity of Microglial/Macrophate Cells
To evaluate the effects of 807A-M0028-B02 on phagocytosis activity, a phagocytosis assay of CTD immobilized on Avidin-coated FluoSphere fluorescent microspheres (Molecular Probes) was developed.
The biotinylated CTD immobilized on 1.2 μm yellow-green latex FluoSpheres® NeutrAvidinTM labeled microspheres (Molecular Probes Europe BV, Leiden, The Netherlands) were resuspended with different concentrations of IgG converted clone 807A-M0028-B02 diluted in OPTI-MEM medium supplemented with 1% BSA (Tissue culture tested, Invitrogen AB, Sweden) and 2% ITS-X (Gibco, Invitrogen AB, Sweden) serum supplement (CM) and incubated for 30 min at +4° C. THP-1 cells (106 cells ml−1) in CM with or without 2.0% NaN3, the inhibitor of phagocytosis, were added at a ratio of 1:100 (cells:beads). To allow binding to Fc-receptors, cells were synchronised at +4° C. for 20 min and the cell-free beads were removed by low-speed centrifugation (200 g, 10 min, +4° C.). The cell pellets were resuspended in CM and incubated at 37° C. for 40 min in a CO2-incubator. After trypsinisation, the cell suspension was taken in sterile conditions, layered over a 7.5% bovine serum albumin (BSA) cushion and centrifuged at 150×g for 10 min at +4° C., to remove non-internalised beads. The cell pellets were resuspended in 0.3 ml of 2% PFA in PBS. The results were expressed as the percent of the control, i.e. amount of phagocytic cells containing two or more beads in presence vs. in absence of antibodies, as determined by flow cytometry. A FACScanTM (Becton Dickinson, San Jose, Calif.) with an air-cooled argon laser providing an excitation at 488 nm was used. A total of 10000 events were acquired for each sample and stored in the list mode data format. The fluorescence emission was collected at 520 nm (FL1) for the phagocytosis. Data collection and analysis were performed with a Consort 30 system and LYSIS-II program. The data were analyzed, once displayed as two-parameter complexity and cell size, in the process of gating and as fluorescence (FL1) frequency distribution histogram to analyze the phagocytosis. EC50, the concentration that induced 50% increase of phagocytic activity was determined for each antibody tested using dose-response curves built with the percentages of phagocytic activity, versus the range of concentrations (0.01-5 μg/ml). Then, the EC50 were extrapolated from these curves and used to compare the relative efficiency of phagocytic stimulation of the antibodies. The IgG converted clone 807A-M0028-B02 demonstrated high efficiency (EC50=34±15 ng/ml) to stimulate THP-1 cells. The results thus indicate that 807A-M0028-B02 specifically directed the in vitro phagocytic uptake of CTD-bearing beads by human macrophage/microglia-like cells in a concentration-dependent fashion.
Example 33
Germ Line Correction of Clones Found in Selections Described in Example 4, Example 21 and Example 22
Of the antibodies described in Table 18, five of them (807A-M0028-B02, 807B-M0004-H03, 807B-M0009-F06, 807B-M0004-A03 and 807B-M0079-D10) have been investigated further. Somatic mutations in the variable part of the light chains of these antibodies have been found in all clones. Some of the clones also contained mutations in the constant part of the light chain (Table 21). Sequence alignments with genomic and known germline sequences have been performed, and the correct amino acids have been identified (indicated in bold in Table 19 and Table 20). The VL chains of the corrected clones are described in Table 19, and the constant parts of the IgG are described in Table 20.
To ensure that the IgG molecules are germline, the somatic mutations are corrected at the DNA level in the five antibodies, and all five germline-corrected IgG1s were expressed (transiently in HEK 293T cells). Comparative binding analysis was performed in Biacore (Example 34), CTD-ELISA and IHC to ensure that the germline-corrected antibodies are still functional. The results are summarised in Table 23.
Example 34
Biacore Analysis of Germ Line Corrected IgGs
The germline-corrected clones described in Tables 19 and 20 were analysed in Biacore. The analysis was performed by running the IgGs at different concentrations over a surface with coated bCTD. A surface with a biotinylated control IgG was used as a negative surface.
Biacore analysis of clones 807A-M0028-B02, 807A-M0028-B02.1 and 807A-M0028-B02.2 showed that the three IgG molecules bind to bCTD with similar kinetics (similar on-rate and off-rate). The affinity of the IgG is not significantly altered. The same results were obtained when comparing 807B-M0004-A03 with 807B-M0004-A03.1.
Biacore analysis of 807B-M0004-H03 and 807B-M0004-H03.1 indicated that 807B-M0004-H03.1 binds with a different kinetic to the parental clone. However, this did not influence the affinity value significantly. The clone 807B-M0009-F06.1 had lost its binding capacity to bCTD as shown in CTD-ELISA and Biacore analysis and CTD-ELISA.
Example 35
Binding of Antibodies to ApoE-CTD by ELISA
Antibodies were screened for ApoE-CTD binding capacity using a coated ApoE-CTD ELISA. Human, marmoset or murine ApoE-CTD was coated on a microtiter plate followed by incubation with test antibodies. After this, the amount of antibody bound was determined by detection with secondary HRP-antibody and tetramethylbenzidine (TMB) substrate. ApoE-CTD binding gives a high signal measured as optical density (OD) in the ELISA. Binding of 807A-M0028-B02, 807A-M0028-B02.1 and 807A-M0028-B02.2 are exemplified in FIG. 22. Results are disclosed in Table 22.
Example 36
Binding of Antibodies to Human Lipoproteins
Antibodies were screened for lipoprotein binding capacity using a coated VLDL ELISA. Human VLDL was coated on a microtiter plate followed by incubation with test antibodies. After this, the amount of antibody bound was determined by detection with secondary HRP-antibody and tetramethylbenzidine (TMB) substrate. VLDL binding gives a high signal measured as optical density (OD) in the ELISA. Binding of 807A-M0028-B02, 807A-M0028-B02.1, 807A-M0028-B02.2, 807B-M0004-H03.0, 807B-M0004-H03.1, 807B-M0004-A03 and 807B-M0004-A03.1 are exemplified in FIG. 23. Results are disclosed in Table 22.
Example 37
In Vivo Binding of 807A-M0028-B02, 807B-M0004H03, 807B-M0004-A03, 807B-M0079-D110807B-M0009-F06 to Mouse Cerebral Plaques and In Vitro Binding of Fab Clones to Human AD Cerebral Plaques
In vivo binding of 807A-M0028-B02, B807B-M0004H03, 807B-M0004-A03, 807B-M0079-D10 and 807B-M0009-F06 to cerebral plaques (immunodecoration) was demonstrated by i.p. or i.v. injections into APP/PS1 transgenic mice (FIGS. 24 and 25). Immunodecoration was observed already two days after a single dose administration of 10 mg/kg of 807A-M0028-B02. Binding of 807A-M0028-B02, B807B-M0004H03, 807B-M0004-A03, 807B-M0079-D10 and 807B-M0009-F06 was only observed on plaques while no staining of astrocytes or any other brain structure was detected (FIGS. 24 and 25). It was demonstrated that a substantial number of plaques were immunodecorated with respective clones by comparing the total plaque burden in each mouse that was determined by staining with a monoclonal antibody to Aβ (6E10) on adjacent sections.
In vitro binding of affinity maturated clones to amyloid plaques in human AD brain sections was also demonstrated by Immunohistochemistry (FIG. 25)
In vitro binding of different wild type clones in human AD plaques is visualised in FIG. 26.
Example 38
Affinity Maturation of 807A-M0028-B02, 807B-M0004H03, 807B-M0004-A03, 807B-M0079-D10807B-M0009-F06 by VH-CDR3 Spiking
Spiking mutagenesis was used to introduce low level mutations over the fall length of the VH-CDR3 of each of 807A-M0028-B02, 807B-M0004H03, 807B-M0004-A03, 807B-M0079-D10 and 807B-M0009-F06 in the context of the original wild-type residues (see FIG. 29). PCR was carried out using an oligionucleotide carrying a region of spiked diversity over the length of the VH-CDR3 sequence of each antibody bracketed by regions of homology with the target V gene in the FR3 and FR4 regions together with a specific primer homologous to the FR1 region capable of annealing to the 5′ end of the target V gene.
For antibodies 807A-M0028-B02, 807B-M0009-F06 and 807B-M0004-H03, diversification of the VH-CDR3 was realized through a one step PCR amplification. This PCR was performed using a 5′ primer complementary to the light chain constant region and the 3′specific spiked oligonucleotides. The PCRs were then performed in a volume of 50 μl using the advantage 2 PCR enzyme system (Clontech) and 10 pmoles of each primer for 25 cycles (1 min at 95° C., 1 min at 60° C., and 2 min at 68° C.). A primer concentration of 100 nM was chosen in order to cover the entire diversity carried by the spiked oligonucleotides. 100 to 200 reactions were needed to obtain  ̃6 μg of PCR products. All products were purified using the GFX purification kit (Amersham).
The resulting PCR products of 730 bp contain an internal XbaI site and an BstEII site, incorporated in the oligonucleotides. These sites were used to clone the products into a display vector.
Diversification of the VH CDR3 of 807B-M04-A03 and 807B-M0079-D10 was realized in two steps: after primary amplification as described above, the resulting PCR products of 730 bp were re-amplified with a combination of a 5′ end nested forward primer, appended with a SfiI site, and a 3′end Nhe1-tagged CH1 reverse primer. The PCRs were then performed in a volume of 50 μl using the advantage 2 PCR enzyme system (Clontech) and 10 pmoles of each primer for 20 cycles (1 min at 95° C., 3 min at 68° C.); 100 to 200 reactions were needed to obtain  ̃6 μg of PCR products. The number of cycles was kept quite low (20 cycles) in order to maintain maximal diversity, introduced in the first PCR step. To ensure again maximal diversity, for each reaction, 50 ng of the first PCR product was used as template to initiate the second PCR reaction. All products were purified using the GFX purification kit (Amersham).
The PCR products and vector backbones were digested using 50 U/μg DNA of either XbaI and BstEII (for 807A-M0028-B02, 807B-M0009-F06 and 807B-M0004-H03) or SfiI and NheI (for 807B-M0004-A03 and 807B-M79-D10).
The resulting cleaved products (both vectors and PCR fragments) were gelpurified, 1.6 μg of each DNA fragment was ligated into 10 μg of similarly cut phagemid vector backbone using T4 DNA ligase (NEB) and the ligation mixture for each spiked library was introduced into E. coli TG1 cells by electroporation.
Phagemid particles were rescued from the libraries using helper phage M13-K07 (Marks et al., (J. Mol. Biol. 222, 581 (1991)) using enough bacteria from each library for inoculation in order to represent each clone at least once.
The diversity in the VH-CDR3 of each library was evaluated by sequencing. 96 isolates were randomly picked for each library and the VH-CDR3 regions were sequenced and compared to the reference wild type VH-CDR3 sequences.
For clones of 807B-M0079-D10 and 807B-M0004-A03 the full VH sequence was determined and compared to the VH reference regions outside VH-CDR3.
A selection procedure consisting of two rounds of selection was used to preferentially enrich the higher affinity clones in the library over the lower affinity clones. The first round of affinity selection was carried out using a reduced antigen concentration relative to the concentration used to select the wild-type antibody. The optimum reduced antibody concentration was determined empirically using the wild-type antibody and a control antibody. The second round was performed at a further reduced antigen concentration in the presence of competing soluble Fab or IgG. The selection conditions used are detailed in Table 3.
To determine if enrichment of antigen positive clones had occurred, 46 randomly picked clones from before and after the first round of selection for each antibody to be matured were tested in an antigen ELISA. In all cases, enrichment of antigen positive clones after just one round of selection was observed.
After selection, the geneIII stump was removed from the vector to allow soluble Fab expression. 200 clones were randomly picked, screened by ELISA and their heavy chains sequenced. The Dyax WEBPHAGE database was used to link ELISA data to the respective sequences. The VH-CDR3 sequences of the clones found to be positive in the screening assay (OD signal=3× background) were analysed further.
For the clones 807B-M0079-D10 and 807B-M0004-A3 the full VH region was amplified and cloned and so the full VH sequence was obtained and compared to the VH reference sequence in order to check for any mutations outside VH CDR3. Clones containing mutations in framework regions were discarded but clones with mutations in the VH-CDR1-CDR2 were kept.
The results of amino acid frequency analysis of selected clones are presented in Tables 24 to 30. In the VH-CDR3, some amino acid positions are very conserved whilst others are frequently mutated.
Biacore screening was used to select five CDR3-mutated Fabs based on off rate or KD. CDR3-mutated Fabs were expressed in bacteria Periplasmic extracts were prepared and screened in Biacore. The best clones were selected based on either off-rates or KD for binding to hCTD or a peptide as shown in Table 4.
The biotinylated peptides or human CTD were coated on streptavidin chips. Periplasmic extracts from 10 ml cultures were diluted 1⁄2 in HBS+0.1% BSA. Samples were injected at 30 μl/min for 3 minutes using the kinject program. Following a 3 minute dissociation, any remaining sample was stripped from chip surface. Off rates were measured on a time window of 1 min (between 10-70s). These data are presented in Tables 31 to 35.
On rates can be calculated from Biacore curves if both koff and the Fab concentration are known. Under conditions of full Mass Transfer Limitation (MLT) like those encountered when working with very high density chips and low flow rates, the Biacore signal depends only on the concentration of the analyte run over the chip surface. Fab concentrations in crude samples can be determined from a standard curve obtained by running different concentrations of a purified Fab on a high density Protein A chip. Using the Fab concentrations obtained that way and the koff values, we calculated kon data from the Biacore curves. The equilibrium dissociation constant KD was obtained from koff/kon These data are presented in Tables 31 to 35. Note that the 807B-M0079-D10 does not bind to Protein A. Therefore, only koff values are presented for this clone.
Five variants were successfully selected for 807B-M0004-A03, 807B-M0004-H03, 807B-M0009-F06 and 807A-M0028-B02. The selected variants are identified in Table 36. No clones with significantly improved Koff were found for 807B-M0079-D10 (no KD data available for this clone).
The selected Fabs were produced in E. coli and purified from periplasmic extracts by Immbolized Metal Affinity Chromatography. The quality of the preparations was checked on reducing and non reducing SDS-PAGE.
The purified Fabs were used to accurately determine the equilibrium dissociation constant KD.
Biotinylated peptides or human CTD were coated on streptavidin chips. Experiments were performed in HBS running buffer. Purified Fabs were diluted to 200 nM and serial 1⁄2 dilutions to 12.5 nM were made and run in duplicates. For association, samples were injected at 30-40 μl/min using the kinject program. Following a 3 minute dissociation, any remaining sample was stripped from the chip surface. Sensorgrams were analyzed using the simultaneous ka/kd fitting program in the BIAevaluation software 3.1. The data are summarized in Table 36. The best clones selected from Biacore screening exhibit an affinity 2 to 3-fold higher than the original clone for 807B-M0004A03, 807B-M0009-F06 and 807A-M0028-B02. In the case of 807B-M0004-H03, the Biacore signals did not allow an accurate comparison of the different clones.
Purified Fabs were also tested by immunohistochemistry. Table 37 shows the names of the chosen clones together with the wild type clones and indicates whether they stain plaques in immunohistochemistry.
Example 39
Light Chain Shuffling of the Antibody Variants Selected from Example 38
As a starting point for light chain shuffling (cycle 2 in FIG. 29), the heavy chains corresponding to the VH-CDR3 improved variants from Example 38 (cycle 1 in FIG. 29) were used together with wild-type (WT) clones.
The WT clone 807B-M0009-F06 was not included because the affinity of this clone was significantly lower compared to the selected variants from cycle 1. For antibody 807B-M0079-D10, as no improved affinity variant was found in cycle 1, the LC shuffling was performed on the WT clone alone.
In this Example, the non-affinity matured antibodies have been designated as the wild type (WT) clone and the selected variants from cycle 1 as parental clones.
The selected heavy chain variants from cycle 1 were cloned into the FAB310 vector backbone containing a repertoire of 5 to 6 heaving chains (HC) shuffled with approximately 108 light chains (LC) to create combinatorial diversity.
For every clone, a Qiagen DNA preparation was performed on a TG1 culture. 10 μg of DNA was then cleaved using SfiI and NotI restriction enzymes, generating a heavy chain fragment size of 650 bp. The FAB310 vector backbone was similarly cut.
The resulting cleaved products (both vector and fragments) were gelpurified and, for each library, the different heavy chain variant fragments were pooled in equal amounts and 3 μg of the pooled fragments were ligated with 6 μg of cleaved phagemid vector backbone using T4 DNA ligase (NEB). Desalted ligation mixtures for each library were introduced into E. coli TG1 cells by electroporation.
The library sizes achieved were such that each heavy chain variant was combined with at least one copy of each member of the light chain repertoire.
Heavy chain sequences were determined for 50 isolates, randomly picked from each library.
The light chain sequences of 48 isolates randomly picked from the 807A-M0028-B02-derived unselected library and 48 from the 807B-M0009-F06 derived unselected library. 63 unique functional light chains were obtained.
Phagemid particles were rescued with helper phage M13-KO7 (Marks et al. J. Mol. Biol. 222, 581 (1991)) using enough bacteria from each library for inoculation in order to represent each clone at least once.
The 5 light chained shuffled libraries were selected for improved affinity variants. Prior to selections the libraries were depleted for streptavidin binding antibodies by pre-incubating the libraries with 100 μl streptavidin paramagnetic beads in 1 ml 2% MPBS. For each library three concentrations of antigen were used to determine the optimal concentration for the second round of selection (Table 5). The incubation time of the phage with bead-target complex was reduced to 0.5 hour and 11 cycles of programmed washing was used in the Kingfisher device. After selection the bound phage were eluted and infected with E. coli (TG1 OD of 0.5) and the liquid amplified overnight at 30° C. with shaking at 250 rpm in 25 ml 2×TY/Ampiclillin (100 ug/ml) Glucose (2% w/v). Cells were concentrated and glycerol stocks were made in order to perform the round 2 selection.
Unselected library and output was titrated to get single colonies for picking and screening. From the unselected library and output library 47 colonies were picked and screened in a phage ELISA (Coated antigen 0.5 μg/ml for all antigens via b-BSA plus streptavidin).
All selection arms resulted in the enrichment of antigen binding clones. Based on these results an antigen concentration was chosen for the second round of selection.
The conditions for the round 2 selection were chosen to be more stringent and were designed to select for improved (faster) kon and improved (slower) koff. Three strategies were used as outlined in Table 6: Strategy I—further lowering of antigen concentrations; strategy II—further lowering of antigen concentration and reduced incubation time with antigen (koff selection); strategy III—further lowering of antigen concentration and increased stringency washing (koff selection). Selection was performed in a KingFisher automated device and an input of approximately 1012 phages were used. Prior to selections the libraries were depleted for streptavidin binding antibodies as described above. After selection the bound phage were eluted and used to infect E. coli as described previously. Cells were concentrated and glycerol stocks were made.
The output of round 2 was pre-screened in ELISA to determine the percentage of antigen binding clones. Sequence analysis of a limited number of clones was performed to determine if any particular clone was dominating selection and if there are any dominant light chain families in the selected clones.
Each of the round 2 phage outputs (15 in total) were re-cloned in batch in order to produce soluble Fabs. This was achieved through the removal of the geneIII stump from the vector.
200 clones were randomly picked and screened by ELISA for their binding to their respective antigen. Antibody sequences were determined for the positive hits only. The storage and initial sequence analysis was conducted via Dyax WEBPHAGE database.
For libraries 807B-M0004-A03, 807B-M079-D10 and 807A-M0004-H03, antibodies enriched between 2 to 8 times were selected. For libraries 807A-M0028-B02 and 807B-M009-F06, all clones were taken since number of unique clones was low. The light and heavy chain sequences from all ELISA positive and unique hits are shown in Tables 38 to 42.
After Biacore analysis of the potential affinity matured binders, a few were found to have a higher affinity. Only libraries 807B-M0004-A03, 807A-M0028-B02 and 807B-M004-H03 gave affinity matured antibodies with a higher affinity than the WT and parental clones. The light chain sequence of those selected clones was aligned to the germline. Interestingly, the same amino acid positions seem to be diversified among all the selected clones belonging to the same germline. The same amino acid positions seem to be diversified among all the selected clones belonging to the same germline.
For library 807B-M0004-A03, although clones M148E08 and M150E03 have exactly the same heavy chain sequence and light chains which differ by two amino acids, clone M150E03 has a 3.2× improved affinity compared to M148E08, suggesting that only two amino acids located in FR1 are responsible for this improvement.
For libraries 807B-M0028-B02 and 807B-M0004-A03, most of the diversification observed in the CDR and FR was reversion to the germline sequence compared to wild-type.
Only a few conservative variations were observed in FR3 of 807A-M0028 B02 library-derived clones.
For Biacore analysis, all clone variants were grown in small cultures (typically 10 ml), periplasmic extracts (PE) were prepared and the Fab concentration in the PEs was measured by running the samples on a Protein A/G chip. The PEs were then diluted to the same Fab concentration (25-50 nM) and run over a target-coated chip (peptide 4, 8, 9 or CTD). The best clones were identified based either on the amplitude of the association and dissociation phases (807A-M0028-B02, 807B-M0004-A03, 807B-M0009-F06) or on the value of the Biacore signal at equilibrium (807B-M0004-H03). Clones derived from 807B-M0079-D10 do not bind to Protein A/G chips and were thus ranked only based on their off-rates
Clones derived from 807B-M0004-H03 were ranked based on the value of the Biacore signal at equilibrium which reflects the equilibrium dissociation constant KD: the higher the Biacore signal at equilibrium, the better the affinity.
Clones derived from 807B-M0079-D10 were ranked based only on koff.
Four and five variants were selected for 807A-M0028-B02 and 807B-M0004-A03, respectively. None of the isolated 807B-M0009-F06 variants seemed to exhibit an affinity higher than the best variants obtained from CDR3 spiking. For 807B-M0004-H03, two variants were selected based on the value of the Biacore signal at equilibrium. None of the 807B-M0079-D10 variants could be selected based on off-rate analysis.
The selected clones selected were produced in E. coli and purified from periplasmic extracts. This material was used to measure accurately the equilibrium dissociation constant KD in Biacore. The data are summarized in Table 7. The best clones isolated from light chain shuffling exhibit an affinity  ̃5-times better than the corresponding original wild type clones or the best clones isolated from CDR3 spiking.
The clones selected following light chain shuffling are shown in Tables 43 and 44.







TABLE 1







Off-rate measurement of sFabs originating from selections


on fibrils and ur-bCTD












koff



Initial name
HCDR3
(e−3s−1)
RU













807A-M0027-C11
AVGYGDYGDY
13.30
79.2


807A-M0027-H05
DFFTSYFDH
16.90
182.0


807A-M0026-F11
DLWFGEWDY
28.10
165.8


807A-M0026-H09
DLWFGEWDY
25.10
138.0


807A-M0027-E12
DLWFGEWDY
8.95
419.9


807A-M0028-B12
DLWFGEWDY
18.80
335.5


807A-M0029-G10
DLWFGEWDY
23.80
212.0


807A-M0027-G01
DRGVSLLGAFDI
30.00
231.3


807A-M0028-A07
ESIAVAGVDY
53.60
367.0


807A-M0026-F05
GRGNYDFWSAGYYYYYMDV
>
158.0


807A-M0028-G07
QEVWQWPAQFDS
35.30
131.7


807A-M0027-E11
SLDLDY
40.50
412.3


807A-M0026-G08
SSGIYYGYYMDV
38.80
594.1


807A-M0028-B02
SVLLDY
28.00
454.6


807A-M0028-B06
DRGVSLLGAFDI
13.50
100.3


807A-M0027-D05
EPIWGYYYYGMDV
9.16
377.8
















TABLE 2







Comparison of Fab and IgG binding on CTD-coated


chip for the candidate clones 807A-M0026-F05 (26F5),


807A-M0027-E11 (27E11) and 807A-M0028-B02 (28B2)















ka (1/Ms)
kd (1/s)



Clone
Format
Surface
e5
e−3
KD (nM)















27E11
Fab
HD
9.5
45.5
47.8 ± 8.1 



IgG
HD
7.5
7.4*
9.9 ± 4.1





8.1
2.1#
2.6 ± 1.1




LD
10.1
7.7*
7.6 ± 3.2





11.1
2.1#
1.9 ± 0.8


28B2
Fab
HD
2.5
44.2
179 ± 5.7 



IgG
HD
3.7
4.0
10.8 ± 2.4 




LD
5.2
6.4
12.3 ± 3.7 


26F5
Fab
HD
n.f.
n.f.
n.d.



IgG
HD
0.2
42
2103 ± 785 




LD
n.f.
n.f.
n.d.





*kd measured directly after injection stop.


#kd measured ~50 sec after injection stop.


n.f. no fit


n.d. not determined















TABLE 3





Antibody
Round 1 selection
Round 2 selection







807A-M0028-B02
3 nM bCTD or
0.3 nM bCTD + 5 μM wt Fab



1 tube of fibrils
or 0.3 nM IgG1


807B-M0004-A03
5.7 nM p4
0.057 nM p4 + 5 μM wt Fab




or 0.01 nM IgG1


807B-M0004-H03
5.7 nM p4
0.57 nM p4 + 5 μM wt Fab




or 0.3 nM IgG1


807B-M0009-F06
5.7 nM p9
0.057 nM p9 + 5 μM wt Fab




or 0.3 nM IgG1


807B-M0079-D10
5.7 nM p8
0.057 nM p8 + 5 μM wt Fab




or 0.3 nM IgG1




















TABLE 4









Number of clones



Original clone
KD (nM)
screened









807A-M0028-B02
150 (on hCDT)
72



807B-M0004-A03
 98 (on p4)
39



807B-M0004-H03
200 (on p4)
54



807B-M0009-F06
172 (on p9)
24



807B-M0079-D10
 26 (on p8)
33

















TABLE 5







Percentage of antigen binding clones retrieved from the round 1


selections at decreasing antigen concentrations










Library
Round
Antigen
Ag concentrations















A03
1
P4
57 nM,
5.7 nM,
0.57 nM


Hit rate %


Input


2%
2%
2%


Output


62% 
51% 
83% 


H03
1
P4
570 nM, 
57 nM,
5.7 nM


Hit rate %


Input


2%
0%
0%


Output


72% 
72% 
81% 


F06
1
P9
57 nM,
5.7 nM,
0.57 nM


Hit rate %


Input


0%
0%
0%


Output


6%
4%
2%


D10
1
P8
57 nM,
5.7 nM,
0.57 nM


Hit rate %


Input


0%
0%
0%


Output


34% 
45% 
38% 


B2
1
CTD
300 nM, 
 30 nM,
  3 nM


Hit rate %


Input


2%
2%
2%


Output


28% 
21% 
17% 
















TABLE 6







Conditions used for the second round selection strategies

















Ag conc
Incubation



Strategy
Library
Round
Antigen
(nM)
time (mins)
Washing
















I
A3
2
Pep-4
0.057
30
11 × 5 mins


I
H3
2
Pep-4
5.7
30
11 × 5 mins


I
D10
2
Pep-8
0.057
30
11 × 5 mins


I
B2
2
b-CTD
10
30
11 × 5 mins


I
F6
2
Pep-9
5.7
30
11 × 5 mins


II
A3
2
Pep-4
0.57
3
11 × 5 mins


II
H3
2
Pep-4
5.7
3
11 × 5 mins


II
D10
2
Pep-8
0.57
3
11 × 5 mins


II
B2
2
b-CTD
30
3
11 × 5 mins


II
F6
2
Pep-9
5.7
3
11 × 5 mins


III
A3
2
Pep-4
0.57
30
3 × 5 mins + 3 × 45 mins + 3 × 5 mins


III
H3
2
Pep-4
5.7
30
3 × 5 mins + 3 × 45 mins + 3 × 5 mins


III
D10
2
Pep-8
0.57
30
3 × 5 mins + 3 × 45 mins + 3 × 5 mins


III
B2
2
b-CTD
30
30
3 × 5 mins + 3 × 45 mins + 3 × 5 mins


III
F6
2
Pep-9
5.7
30
3 × 5 mins + 3 × 45 mins + 3 × 5 mins




















TABLE 7





Clone name
kon (1/Ms)
koff (1/s)
KD (nM)







M0004A03 WT
4.19E+05
3.31E−02
79
*


M0004A03-M0148-E08
9.51E+05
4.85E−02
51
*


M0004A03-M0149-F02
6.51E+05
2.41E−02
37
*


M0004A03-M0149-G11
4.12E+05
1.73E−02
42
*


M0004A03-M0150-E03
6.08E+05
9.89E−03
16
*


M0004A03-M0151-D09
5.35E+05
1.61E−02
30
*


M0028B02-M0168-D10
6.97E+04
2.97E−03
43
*


M0028B02-M0169-F03
1.73E+05
1.78E−03
10
*


M0028B02-M0171-E03
1.02E+05
2.61E−03
26
*


M0028B02-M0171-G02
8.99E+04
4.55E−03
51
*





* Kinetic analysis, 1:1 model













TABLE 8





Description of SEQ ID NOS: 21-164 and 171-206




















Antibody







Name
VH-CDR1
VH-CDR2
VH-CDR3
VH
VL-CDR1





807A-M0027-E11
SEQ ID NO: 21
SEQ ID NO: 22
SEQ ID NO: 23
SEQ ID NO: 39
SEQ ID NO: 30


807A-M0028-B02
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 40
SEQ ID NO: 33


807A-M0026-F05
SEQ ID NO: 27
SEQ ID NO: 28
SEQ ID NO: 29
SEQ ID NO: 41
SEQ ID NO: 36


807B-M0001-B07
SEQ ID NO: 45
SEQ ID NO: 46
SEQ ID NO: 47
SEQ ID NO: 135
SEQ ID NO: 90


807B-M0004-A03
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 50
SEQ ID NO: 136
SEQ ID NO: 93


807B-M0004-A05
SEQ ID NO: 51
SEQ ID NO: 52
SEQ ID NO: 53
SEQ ID NO: 137
SEQ ID NO: 96


807B-M0004-C04
SEQ ID NO: 54
SEQ ID NO: 55
SEQ ID NO: 56
SEQ ID NO: 138
SEQ ID NO: 99


807B-M0004-C05
SEQ ID NO: 57
SEQ ID NO: 58
SEQ ID NO: 59
SEQ ID NO: 139
SEQ ID NO: 102


807B-M0004-F06
SEQ ID NO: 60
SEQ ID NO: 61
SEQ ID NO: 62
SEQ ID NO: 140
SEQ ID NO: 105


807B-M0004-F10
SEQ ID NO: 63
SEQ ID NO: 64
SEQ ID NO: 65
SEQ ID NO: 141
SEQ ID NO: 108


807B-M0004-H03
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 68
SEQ ID NO: 142
SEQ ID NO: 111


807B-M0009-C03
SEQ ID NO: 69
SEQ ID NO: 70
SEQ ID NO: 71
SEQ ID NO: 143
SEQ ID NO: 114


807B-M0009-F06
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 74
SEQ ID NO: 144
SEQ ID NO: 117


807B-M0013-A12
SEQ ID NO: 75
SEQ ID NO: 76
SEQ ID NO: 77
SEQ ID NO: 145
SEQ ID NO: 120


807B-M0079-D10
SEQ ID NO: 78
SEQ ID NO: 79
SEQ ID NO: 80
SEQ ID NO: 146
SEQ ID NO: 123


807B-M0081-F12
SEQ ID NO: 81
SEQ ID NO: 82
SEQ ID NO: 83
SEQ ID NO: 147
SEQ ID NO: 126


807B-M0081-H03
SEQ ID NO: 84
SEQ ID NO: 85
SEQ ID NO: 86
SEQ ID NO: 148
SEQ ID NO: 129


807B-M0083-E11
SEQ ID NO: 87
SEQ ID NO: 88
SEQ ID NO: 89
SEQ ID NO: 149
SEQ ID NO: 132





Antibody



Polynucleotide
Polynucleotide


Name
VL-CDR2
VL-CDR3
VL
encoding VH
encoding VL





807A-M0027-E11
SEQ ID NO: 31
SEQ ID NO: 32
SEQ ID NO: 42
SEQ ID NO: 174
SEQ ID NO: 173


807A-M0028-B02
SEQ ID NO: 34
SEQ ID NO: 35
SEQ ID NO: 43
SEQ ID NO: 176
SEQ ID NO: 175


807A-M0026-F05
SEQ ID NO: 37
SEQ ID NO: 38
SEQ ID NO: 44
SEQ ID NO: 172
SEQ ID NO: 171


807B-M0001-B07
SEQ ID NO: 91
SEQ ID NO: 92
SEQ ID NO: 150
SEQ ID NO: 178
SEQ ID NO: 177


807B-M0004-A03
SEQ ID NO: 94
SEQ ID NO: 95
SEQ ID NO: 151
SEQ ID NO: 180
SEQ ID NO: 179


807B-M0004-A05
SEQ ID NO: 97
SEQ ID NO: 98
SEQ ID NO: 152
SEQ ID NO: 182
SEQ ID NO: 181


807B-M0004-C04
SEQ ID NO: 100
SEQ ID NO: 101
SEQ ID NO: 153
SEQ ID NO: 184
SEQ ID NO: 183


807B-M0004-C05
SEQ ID NO: 103
SEQ ID NO: 104
SEQ ID NO: 154
SEQ ID NO: 186
SEQ ID NO: 185


807B-M0004-F06
SEQ ID NO: 106
SEQ ID NO: 107
SEQ ID NO: 155
SEQ ID NO: 188
SEQ ID NO: 187


807B-M0004-F10
SEQ ID NO: 109
SEQ ID NO: 110
SEQ ID NO: 156
SEQ ID NO: 190
SEQ ID NO: 189


807B-M0004-H03
SEQ ID NO: 112
SEQ ID NO: 113
SEQ ID NO: 157
SEQ ID NO: 192
SEQ ID NO: 191


807B-M0009-C03
SEQ ID NO: 115
SEQ ID NO: 116
SEQ ID NO: 158
SEQ ID NO: 194
SEQ ID NO: 193


807B-M0009-F06
SEQ ID NO: 118
SEQ ID NO: 119
SEQ ID NO: 159
SEQ ID NO: 196
SEQ ID NO: 195


807B-M0013-A12
SEQ ID NO: 121
SEQ ID NO: 122
SEQ ID NO: 160
SEQ ID NO: 198
SEQ ID NO: 197


807B-M0079-D10
SEQ ID NO: 124
SEQ ID NO: 125
SEQ ID NO: 161
SEQ ID NO: 200
SEQ ID NO: 199


807B-M0081-F12
SEQ ID NO: 127
SEQ ID NO: 128
SEQ ID NO: 162
SEQ ID NO: 206
SEQ ID NO: 205


807B-M0081-H03
SEQ ID NO: 130
SEQ ID NO: 131
SEQ ID NO: 163
SEQ ID NO: 202
SEQ ID NO: 201


807B-M0083-E11
SEQ ID NO: 133
SEQ ID NO: 134
SEQ ID NO: 164
SEQ ID NO: 204
SEQ ID NO: 203
















TABLE 9





Amino acid sequences of the VL chains of the antibodies identified using the screening strategy of Example 5



















Initial Name
LV-FR1
LV-CDR1
LV-FR2
LV-CDR2





807A-M0027-C11
QDIQMTQSPSTLSASVGD
RASQSVSSWLA
WYQQKPGAAPRLLIY
KASSLQT



RVTITC


807A-M0043-F08
QDIQMTQSPSSVSASVGD
RASQGISSWLA
WYQQKPGKAPKLLIY
AASSLQS



RVTITC


807A-M0039-E07
QSELTQPPSASGSPGQSV
TGTSSDVGGYNYVS
WYQQHPGKAPKLMIY
EVSKRPS



TISC


807A-M0039-D11
QDIQMTQSPSSLSASVGD
QASQDIRNYIN
WYQQKPGKAPKLLIN
DASNLEP



RVTMTC


807A-M0037-F10
QSALTQPPSASGTPGQRV
SGRSSNIGSNSVN
WYQQLPGTAPKLLIY
SNNQRPS



TISC


807A-M0028-B06
QDIQMTQSPDTLSLSPGDR
RASQSVSSNYLA
WYHQKPGQAPRLVIY
NTSRRAT



ATLSC


807A-M0046-F05
QDIQMTQSPGTLSLSPGER
RASQSVSSSYLA
WYQQKPGQTPRLLIY
GASSRAT



ATLSC


807A-M0041-F03
QDIQMTQSPSTLSASVGD
RASQGINRWLA
WYQQKPGKAPKLLIY
KASALES



RVAITC


807A-M0043-E08
QYELTQPPSASGSPGQSV
TGTSSDVGAYNYVS
WYQQHPGKAPKLIIY
EVNKRPS



TISC


807A-M0042-D05
QDIQMTQSPATLSVSPGD
RASQSVGSTLA
WYQQKPGQAPRLLIY
GAVTRAT



RVTLSC


807A-M0029-G10
QDIQMTQSPSSLSASVGD
RASQSISSYLN
WYQQKPGKAPKLLIY
AASSLQS



RVTITC


807A-M0046-G03
QDIQMTQSPSSLSASVGD
RASQGITNWVA
WYQQKPGKAPKLLIY
GASRLQS



RVTITC


807A-M0037-D06
QDIQMTQSPSSLSASVGD
RTSQDVRNWVA
WYQQKPGKAPNLLIY
MASTLQS



RVTITC


807A-M0043-E07
QDIQMTQSPSSLSASVGD
RASQNVNTFLN
WYQHKAGKAPKLLIY
AASSLQS



RVTITC


807A-M0027-E11
QDIQMTQSPSSLSASVGD
RASQRIRKNLH
WYQQKPGKAPNLLIY
DASSNER



RVTITC


807A-M0046-A11
QDIQMTQSPSSLSASVGD
RASQSISSYLN
WYQQKPGKAPKLLIY
AASSLQS



RVTITC


807A-M0041-E01
QDIQMTQSPSSLSASVGD
HASQDIANYLS
WYQQKPGKAPKLLIY
DAFNLET



RVTITC


807A-M0044-B07
QDIQMTQSPGILSLSPGER
RASQNLIFNFLA
WYQHKPGQAPRLLIY
GSSTRAT



ATLSC


807A-M0028-B02
QDIQMTQSPSSLSASVGD
RTSQDIRNHLG
WFQQKPGKAPQRLIR
EASILQS



RVTITC


807A-M0039-E06
QDIQMTQSPSSVSASVGD
RASQGISSWLA
WYQQKPGKAPKLLIY
AASSLQS



RVTITC


807A-M0040-A03
QSELTQPPSVSVSPGQTA
SGDKLGDKYAS
WYQQRPGQSPVLVIY
QDTKRPS



TITC


807A-M0044-G07
QDIQMTQSPSSVSASVGD
RASQVISTWLS
WYQQKPGKAPKLLIY
TASTLQS



RVTITC


807A-M0044-E08
QSPSSVSTSVGDRVTITCR
LDIQMTDISTWLA
WYQQKPGKAPKLLIY
AASTLES



ASQ


807A-M0038-A09
QSALTQPPSVSVAPGQTA
GGNNIGTKIVN
WYQQRPGQAPVVVVY
DNSDRPS



RITC


807A-M0037-C08
QDIQMTQSPSSVSASVGD
RASQGISSWLA
WYQQKPGKAPKLLIY
AASSLQS



RVTITC


807A-M0039-H09
QDIQMTQSPSSLPASVGD
RTSQSISDYVN
WYQQKPGKAPNLLIY
AASTLQG



SVTVTC


807A-M0039-D05
QDIQMTQSPSSLSASVGD
RASQDIRDDLG
WYQQKPGKAPKRLIY
AASSLQS



RVTITC


807A-M0042-F12
QSELTQPPSASGTPGQRV
SGGYSNMGSNYAH
WYQQLPGTAPKLLIY
NNNQRPS



TISC


807A-M0043-H05
QSELTQPASVSGSPGQSIT
TGTNTDVGGYNYVA
WYQQHPGKAPKLMIY
DVSNRPS



ISC


807A-M0042-C03
QDIQMTQSPATLSLSPGER
RASQSVSSSYLA
WYQQKPGQAPRLLIY
GASSRAT



ATLSC


807A-M0040-C03
QDIQMTQSPGTLSLSPGER
RASQIFSSSYVA
WYQQKPGQAPRLLIY
GASSRAS



ATLSC


807A-M0046-C05
QDIQMTQSPSSLSASVGD
RASQSISSYLN
WYQQKPGKAPKVLIY
GTSSLQS



RVTITC


807A-M0027-D05
QDIQMTQSPSSLSASVGD
RASQSISSYLN
WYQQKPGKAPKLLIY
AASSLQS



RVTITC


807A-M0040-B11
QSVLTQPPSASGTPGQRV
SGSSSNIGSNNVN
WYQQLPGTAPKLLIY
SNDQRPS



TISC


807A-M0039-B02
QDIQMTQSPSTLSASVGD
RASQSISSWLA
WYQQKPGKAPKLLIY
TASSLES



RVTITC


807A-M0041-C07
QDIQMTQSPSSLSASLGDR
RASQGISNSLA
WYQQKPGKAPKLLIS
AASTLQT



VTITC


807A-M0041-H04
QSELTQPPSASGTPGQRVI
SGSGSNIGSNIVS
WFQQLPGAAPRLLIY
NDHRRPS



ISC


807A-M0028-G07
QYELTQPPSVSVAPGQTA
GGNNIGSKNVH
WYQQKPGQAPVLVVY
DDTDRPS



RITC


807A-M0041-A09
QDIQMTQSPATLSLSPGER
RASQSVSNNLA
WYQQKPGQAPRLLIS
GASTRAT



ATLSC


807A-M0042-B10
QSALTQPASVSGSPGQSIT
SGTDSDVGGYNHVS
WYQQHPGKAPKLIIY
DVDHRPS



ISC


807A-M0041-E06
QSVLTQPPSTSGTPGQRV
SGSNSNIGSKTVN
WYQQLPGTAPKLLIY
MNYERPS



TISC


807A-M0037-D10
QDIQMTQSPSSLSASVGD
RASQSIYTSLN
WYQQKPGKAPRLLIS
DASNLQS



RVTITC


807A-M0044-F04
QDIQMTQSPATLSVSPGG
RASQSVRKNVA
WYQQKPGQPPRLLIY
GASTRAT



RATLSC


807A-M0043-D10
QDIQMTQSPATLSVSPGE
RASQSVSSGLA
WYQQKPGQSPRLLIY
DISTRAT



GATLSC


807A-M0043-G06
QDIQMTQSPSYLSASVGD
RASQTISRYLN
WYQQKPGNAPKLLIY
AASTLQS



RVTITC


807A-M0037-G01
QDIQMTQSPATLSVSPGER
RASQSVSSNLA
WYQQKPGQAPRLLIY
GASTRAT



ATLSC


807A-M0044-E11
QSALTQPASVSGSPGQSIT
TGTSTDVGGYNYVS
WYQKHPGKAPKLMIY
DVSNRPS



ISC


807A-M0043-A10
QDIQMTQSPSSLSASVGD
RASQSISSYLN
WYQQKPGKAPKLLIY
TTSFVQS



RVTITC


807A-M0045-B03
QYELTQPASVSGSPGQSIT
TGTSSDVGAFNYVS
WYQHHPGKAPKLLLY
EVTNRPS



ISC


807A-M0038-A08
QDIQMTQSPSSLSASVGD
RASQSIRIYLN
WYQQKPGKAPKLLIY
AASKLED



RVTITC


807A-M0039-C02
QDIQMTQSPGTLSLSPGD
RASQSVGSDYLA
WYQQKPGQAPRLLIF
AASTRAT



RATLSC


807A-M0027-G01
QSELTQPPSASGTPGQRV
SGGYSNMGSNYAH
WYQQLPGTAPKLLIY
NNNQRPS



TISC


807A-M0039-B08
QDIQMTQSPSSLSASVGD
RASQGISNFLA
WYQQKPGKAPKVLIY
DASTLRS



RVTITC


807A-M0048-E12
QDIQMTQSPSSLSASVGD
RASQGIRNDLG
WYQQKPGKAPKRLIY
GASSLQS



RVTITC


807A-M0041-A08
QSELTQPASVSGSPGQSIT
TGTSSDVGGYNYVS
WYQQHPGKAPKLMIY
DVSNRPS



ISC


807A-M0037-C09
QDIQMTQSPSSLSASVGD
RASQGIRNNLA
WYQQRPGKAPKRLIY
GASNLHS



RVTVTC


807A-M0040-G01
QDIQMTQSPSSLSASVGD
RASQGIHNYVN
WYQQKPGKAPKLLIY
AASSLQS



RVTITC


807A-M0045-E04
QDIQMTQSPSSLSASVGD
RASQGIRKDLG
WYQQRPGKAPKLLIY
GASSLLN



RVTITC


807A-M0041-H05
QSALTQPPSVSGAPGQRV
TGSSSNIGAPYDVH
WYQQVPGTAPKVLIY
GNNHRPS



TISC


807A-M0043-A08
QSALTQPASVSGSPGQSIT
TGTSNDVGGYNSVS
WYQQHPGKAPKLLIY
DVTNRPS



ISC


807A-M0038-C09
QDIQMTQSPDSLAVSLGER
KSSQSVLYSSNNKN
WYQQKPGQPPKLLIY
WASIRES



ATINC
YLA


807A-M0042-F09
QDIQMTQSPSSLSASVGD
RASQGIRHDLG
WYQQKPGKAPKRLIY
AASSLQN



RVTITC


807A-M0045-B12
QDIQMTQSPSSLSASVGD
RTSQNINTYLN
WYQQKPGKAPRLLIY
AASSLQS



RVTITC


807A-M0044-C04
QDIQMTQSPSSLSASVGD
RASQTISNYLN
WYQQKPGKAPKLLIY
ATSTLQS



RVTITC


807A-M0026-F11
QSVLTQPASVSGSPGQSIT
TGTSSDVGIYNYVS
WYQQHPGKAPKLMIY
DVSNRPS



ISC


807A-M0027-E12
QDIQMTQSPGTLSLSPGER
RASRSLFSTYLA
WYQQKPGQPPRLLIY
GASTRAT



ATLSC


807A-M0028-B12
QDIQMTQSPLSLSASAGDR
RASQNINRYLN
WYQQKPGKAPRLLIY
AASNLQS



VTITC


807A-M0026-G08
QDIQMTQSPSSLSASAGD
RANQGIRNNLA
WFQQKPGKAPKSLIY
DASSLQS



RVTITC


807A-M0043-A07
QDIQMTQSPSSVSASVGD
RASQGISSWLA
WYQQKPGKAPKLLIY
AASSLQS



RVTITC


807A-M0042-F04
QDIQMTQSPATLSLSPGES
RASQSVNDYLA
WYQQKPGQAPRLLIY
DSSNRAT



ATLSC


807A-M0045-H09
QDIQMTQSPSSLSASVGD
RASQSISSYLN
WYQQKPGKAPKLLIY
VASSLQS



RVTITC


807A-M0046-D04
QDIQMTQSPGTLSLSPGES
RASQSISSYLN
WYQQKQGKAPKLLMF
AASSLKS



ATLSC


807A-M0040-G04
QDIQMTQSPATLSASVGD
RASQSISSYLN
WYQQKPGKAPKLLIY
AASSLQS



RVTITC


807A-M0045-B01
QSVLTQPASVSGSPGQSIT
TGTNTDVGGYNYVS
WYQQNPGEAPKLIIY
EVNHRPS



ISC


807A-M0040-A08
QSVLTQPPSASGTPGQRVI
SGSSSNIGSNIVS
WFQQVPGAAPRLLIY
NDHRRPS



ISC


807A-M0026-F05
QDIQMTQSPGTLSLSPGER
RASQSIGSRYLA
WYQQKPGQAPRLLIY
DASKRAT



ATLSC


807A-M0037-H02
QDIQMTQSPGTLSLSPGER
RASQSVSSSYLA
WYQQKPGQAPSLLIY
DMSTRAP



ATLSC


807A-M0042-A06
QSALTQPPSASGTPGQRV
SGGYSNMGSNYAH
WYQQLPGTAPKLLIY
NNNQRPS



TISC


807A-M0028-A07
QDIQMTQSPSSLSASIGDR
RASQGISNYLA
WYQQKPGKVPNLLIY
AASTLQS



VTITC


807A-M0043-G01
QDIQMTQSPSTLSASVGD
RPSQSTSNWLA
WYQQKPGKAPKLLIY
KASILES



RVTITC


807A-M0046-F04
QDIQMTQSPSSLSASVGD
RASQSISTYLN
WYQHKPGNAPNLLIY
GASSLKR



RVTITC


807A-M0037-F03
QDIQMTQSPGTLSLSPGER
RASQSISSRYLA
WYQQKAGQAPRLLMY
GASRAT



ATLSC


807A-M0039-C10
QSALTQPRSVSGSPGQSV
TGTYSDVGNYYSVS
WYQQHPGKAPKFIIY
DVTKRPS



TISC















Initial Name
LV-FR3
LV-CDR3
LV-FR4







807A-M0027-C11
GVPSRFSGGGSGTEFTLTISSLQP
QQSYSTPT
FGGGTKVEIK




DDFATYYC



807A-M0043-F08
GVPSRFSGSGSGTDFTLTISSLQP
QQANSFPPT
FGQGTKVEIQ




EDFATYYC



807A-M0039-E07
GVPDRFSGSKSVTSASLAITGLQA
QSYDSSLSGYV
FGSGTKVTVL




EDEADYYC



807A-M0039-D11
GVPSRFRGSGYGTDFSFSISSLQS
QQYDSVPIT
FGQGTRVEIK




EDIATYYC



807A-M0037-F10
GVPDRFSGSKSGTSASLAISGLRS
AAWDDSLSGVV
FGGGTKLTVL




EDEADYYC



807A-M0028-B06
GIPDRFSGSGSGTDFTLTISRLDPE
QQYAYGRSPGYP
FGQGTRLEIK




DFGVYYC



807A-M0046-F05
GIPDRFSGSGSGTDFTLTISRLEPE
QQYGSSPYT
FGQGTKLEIR




DFAVYYC



807A-M0041-F03
GVPSRFSGSVSGTQFTLTISSLQP
QHYYTYPYA
FGQGTKLEIK




DDFATYYC



807A-M0043-E08
GVPDRFSASKSGNTASLTVSGLQ
NSYAGSNSLI
FGGGTKLTVL




AEDEADYYC



807A-M0042-D05
GVPARFSASASGPDFTLTISSLQS
QQYGGSPWYT
FGQGTKLEIK




EDFAVYYC



807A-M0029-G10
GVPSRFSGSGSGTDFTLIISDLQPE
QQSYTTPFT
FGPGTTVDIK




DFATYYC



807A-M0046-G03
GVPSRFSGSGSGTDFSLTISSLQP
QQSYSSLFT
FGPGTKVDIK




EDFATYYC



807A-M0037-D06
GVPSRFSGSGSGTDFTLTISSLQP
QQADTFPWT
FGQGTKVDIK




EDFATYFC



807A-M0043-E07
GVPSRFSGTGSGTDFTLTISSLQP
QQSYSDPLT
FGGGTKVEIK




EDFATYYC



807A-M0027-E11
GVPSRFSGRGSGTEFTLTISSLQP
QQSFSSPWT
FGQGTKVEIK




EDLATYYC



807A-M0046-A11
GVPSRFSGSGSGTDFTLTISSLQP
QQSYSTPLT
FGGGTKVEIK




EDFATYYC



807A-M0041-E01
GVPSRFSGSGSGTDFTLTISSLQP
QQFEDLFSLT
FGPGTRVDLK




EDIATYYC



807A-M0044-B07
GIPDRFSGSGSGTDFTLTINRLEPE
QQYHTSSFT
FGPGTKVDIK




DFAVYYC



807A-M0028-B02
GVPSTFYGSGYGREFTLTISSLQP
LQYDSFPYT
FGQGTKLEIK




EDFATYYC



807A-M0039-E06
GVPSRFSGSGSGTDFTLTISSLQP
QQSYSSPGIT
FGPGTKVEIK




EDFASYFC



807A-M0040-A03
GIPERFSGSNSGNTATLTISGTQT
QAWGSSPVV
FGGGTRLTVL




MDEADYYC



807A-M0044-G07
GVPARFSGSGSGTDFTLTINNLQP
QQANSFPIT
FGGGTKVEIN




EDFATYYC



807A-M0044-E08
GVPSRFSGSGSGTDFTLTISSLQP
QQAYSFPIT
FGQGTRLEIK




EDFATYYC



807A-M0038-A09
GIPERFSGSNSGNTATLTISRVEA
QLWDSSSDHPI
FGTGTKVTVL




GDEADYYC



807A-M0037-C08
GVPSRFSGSASGADFTLTISSLQP
QQTYDTPFT
FGPGTTVDLK




EDFATYYC



807A-M0039-H09
GVPSRFSGSASGTNFSLTIDDLQP
QQTFFSPPT
FGQGTRVEIK




EDFATYYC



807A-M0039-D05
GVPSRFSGSGSGTEFTLTISSLQP
QQHNNYPSFT
FGPGTRLDIK




EDFATYYC



807A-M0042-F12
GVPDRFSGSKSGTSASLAISGLQS
AAWDDSLNGWV
FGGGTKLTVL




EDEADYYC



807A-M0043-H05
GVSTRFSGSKSGDTASLTISGLQT
SSFTSRSTHV
FGTGTKVTVL




EDEADYYC



807A-M0042-C03
GIPDRFSGSGSGTDFTLTISRLEPE
QQSYSSPGIT
FGPGTKVEIK




DFASYFC



807A-M0040-C03
GIPDRFSGSGSGTDFTLTISRLEPE
QQSSSSPPT
FGQGTRVEVR




DFAVYWC



807A-M0046-C05
GVPSRFSGSGSRTDFTLTISSLQP
QQSYNTPPT
FGQGTKLEIK




EDFGIYYC



807A-M0027-D05
GVPSRFSGSGSGTDFTLTISSLQP
QQTYTTPAWT
FGQGTKVEIR




EDFATYYC



807A-M0040-B11
GVPDRFSGSKSATSASLAISGLQS
AAWDDSLNGPV
FGGGTKLTVL




EDEADYHC



807A-M0039-B02
GVPSRFSAGGSGTEFTLTISSLQP
QQYNSYSLT
FGGGTKVEIK




DDFGTYYC



807A-M0041-C07
GVPSRFSGSGSGTDFTLIITNLQPD
QQINGYPVT
FGAGTKVEIK




DFATYYC



807A-M0041-H04
GVPDRFSGSKSGTSASLAITGLRS
AAWDDSLSAVV
FGGGTKLTVL




EDETDYYC



807A-M0028-G07
GIPERFSGSNSGDTATLTISWVEA
HVWDSSSDHYV
FGTGTAVTVL




GDEAKYHC



807A-M0041-A09
GIPARFSGSGSGTEFTLTINSLQSE
QQYDNWPPFT
FGPGTKVDIK




DSAVYYC



807A-M0042-B10
GISNRFSGSKSGNTASLTISGLQA
SSYRSGSTYV
FGTATKVTVL




EDEADYYC



807A-M0041-E06
GVPDRFSGSKSGTSASLAISGLQS
AAWDDSLSGPV
FGGGTKLTVL




EDEADYYC



807A-M0037-D10
GVPSRFSGSGSGTDFTLTIASLQP
QQSYRLFPT
FGQGTRLEIK




DDFATYHC



807A-M0044-F04
GVPARFSGSGSGTEFTLTISRMQP
QQYSSWPA
FGQGTMVEIN




EDFVVYHC



807A-M0043-D10
GIPARFSGSGSGTEFTLTISSLQSE
QQYKDWPLT
FGGGTQVEIK




DFAVYYC



807A-M0043-G06
GVPSRFSGSGSGTDFTLAISSLQP
QNSYSSPYT
FGQGTNVELK




EDFATYYC



807A-M0037-G01
GIPARFSGSGSGTEFTLTISSLQSE
QQYGSSPPIT
FGQGTRLEIK




DFAVYYC



807A-M0044-E11
GVSNRFSGSKSGNTASLTISGLQA
SSYTNTITVV
FGGGTKLTVL




EDEADYYC



807A-M0043-A10
GVPSRFSGSGSGTDFTLTISSLQP
QQSYTIPTT
FGGGTKVDVK




EDFATYYC



807A-M0045-B03
GVSDRFSGSKSGNTASLTISGLQA
ASYTRTRSLA
FGGGTRLTVL




EDEADYHC



807A-M0038-A08
GVPSRFSGSGTGTDFTLTIRSLQP
QQSYSSPGIT
FGPGTKVEIK




EDFASYFC



807A-M0039-C02
GIPDRFSGSGSATDFTLTISSLEPE
QQYASPPRT
FGQGTKVEIK




DFAVYFC



807A-M0027-G01
GVPDRFSGSKSGTSASLAISGLRS
AAWDDSLSGPV
FGGGTKLTVL




EDEADYYC



807A-M0039-B08
GVPSRFSGSGSGTDFTLTIDSLQP
QQYYRYPLT
FGGGTKVEIK




EDFATYYC



807A-M0048-E12
GVPSRFSGSGSGTEFTLTISSLQP
LQHNSYPLT
FGGGTKVEIK




EDFATYYC



807A-M0041-A08
GVSNRFSGSKSGNTASLTISGLQA
SSYTSSSTLDPYA
FGTGTKVTVL




EDEADYYC



807A-M0037-C09
GVPSRFSGSGSGTEFTLTISSLQP
LQHNNYPYS
FGQGTKLEIK




EDFATYYC



807A-M0040-G01
GVPSRFSGSGSATDFTLTISSLQP
QQSFNTPFT
FGPGTRVDIK




EDFATYYC



807A-M0045-E04
GVPSRFSGSGSGTDFTLTISSLQP
LQDNDYPFT
FGPGTKVEIR




EDFATYYC



807A-M0041-H05
GVPDRFSGSKSGTSASLAISGLQA
QSYDSSLSGPI
FGGGTTLTVL




EDEAHYYC



807A-M0043-A08
GVSNRFSTSQSANTASLTISGLQP
SSYTTRSTWV
FGGGTKLTVL




EDEAEYFC



807A-M0038-C09
GVPDRFSGSGSGTDFTLTISSLQA
QQYYSTPTWT
FGQGTKVEIK




EDVAVYYC



807A-M0042-F09
GVPSRFSGSGSGTEFTLTISSLQP
LQHNSYPWT
FGQGTKVEIK




EDFATYYC



807A-M0045-B12
GVPSRFSGSGFGTDFTLTISSLQP
EQSYNVPRT
FGQGTRLDIK




EDFGIYYC



807A-M0044-C04
GVPSRFSGSGSGTDFTLTITSLQP
QQTYNTPGT
FGQGTKLEIK




EDFATYYC



807A-M0026-F11
GVSNRFSGSKSGNTASLTISGLQA
SSYTSSSTPYV
FGTGTKVTVL




EDEADYYC



807A-M0027-E12
GIPDRFSGSGSGTDFTLTISRLEPE
QQYVSSQLT
FGGGTKVEIK




DSALYYC



807A-M0028-B12
GVPSRFSGSQSGTDFTLTISSLQP
QQSFSPPIT
FGQGTRLDIK




EDFATYYC



807A-M0026-G08
GVPSKFSGTGSGTEFTLTIGSLQP
QQYFTFPLT
FGGGTKVEIK




EDSATYYC



807A-M0043-A07
GVPSRFSGSGSGTDFTLTISSLQP
QQANSFPLT
FGGGTKVEIK




EDFATYYC



807A-M0042-F04
GIPARFSGSGSGTDFTLTISSLEPE
QQANSFPPT
FGQGTKVEIK




DFATYYC



807A-M0045-H09
GVPSRFSGSGSGTDFTLTISSLQP
QQSYSIPPT
FGQGTRVEIK




EDFATYYC



807A-M0046-D04
GIPDRFSGRGSGTDFSLTISRLEPE
QQYEFSPEN
FGQGTKLQIK




DFAVYYC



807A-M0040-G04
GVPSRFSGSGSGTDFTLTISSLQP
QQSYSTPHT
FGQGTKLEIK




EDFATYYC



807A-M0045-B01
GVSDRFSGSKSGNTASLTISGLQA
SSYTNRNGYV
FGTGTKVTVL




DDETDYYC



807A-M0040-A08
GVPDRFSGSKSGTSASLAISGLQS
ASWDDSLNGVL
FGGGTKLTVL




EDDADYYC



807A-M0026-F05
GVPVRFSGSGSGTDFTLTISSLGP
QQGYNWPPWT
FGQGTKVEIK




EDFAVYYC



807A-M0037-H02
GIPERFSGSGSGTDFTLTISRLEPE
QQYGSSVA
FGGGTKVEMK




DFAVYYC



807A-M0042-A06
GVPDRFSGSKSGTSASLAISGLRS
AAWDDSLSGPV
FGGGTKLTVL




EDEADYYC



807A-M0028-A07
GVPSRFSGSGSGTDFTLTISSLQP
QKYNSAPRT
FGQGTKVEIK




EDVATYYC



807A-M0043-G01
GVPSRFSGSGSGTEFTLTISSLQP
QQYDSYWT
FGQGTKIEIK




DDFATYYC



807A-M0046-F04
GVPSRFSGSGSETEFTLTISSLQP
QQSYSAPLI
FGGGTKVEIR




EDFATYYC



807A-M0037-F03
GIPARFSGSGSGTDFTLTISSLQPE
QQSYEYPLT
FGQGTKLEIK




DFATYYC



807A-M0039-C10
GVPDRFSGSKSGNTASLTISGLQA
CSYAGSYTLL
FGGGTKLTVL




EDEADYYC

















TABLE 10





Amino acid sequences of the VH chains of the antibodies identified using the screening strategy of Example 5



















Initial Name
FR1
CDR1
FR2
CDR2





807A-M0027-C11
EVQLLESGGGLVQPGG
EYGMS
WVRQAPGKGLEWVS
VISPSGGGTEYADSVKG



SLRLSCAASGFTFS


807A-M0043-F08
EVQLLESGGGLVQPGG
EYTML
WVRQAPGKGLEWVS
GIWPSGGPTFYADSVKG



SLRLSCAASGFTFS


807A-M0039-E07
EVQLLESGGGLVQPGG
FYFMG
WVRQAPGKGLEWVS
SISSSGGMTTYADSVKG



SLRLSCAASGFTFS


807A-M0039-D11
EVQLLESGGGLVQPGG
FYGMG
WVRQAPGKGLEWVS
YISPSGGSTTYADSVKG



SLRLSCAASGFTFS


807A-M0037-F10
EVQLLESGGGLVQPGG
FYGMG
WVRQAPGKGLEWVS
YISSSGGLTFYADSVKG



SLRLSCAASGFTFS


807A-M0028-B06
EVQLLESGGGLVQPGG
FYQMN
WVRQAPGKGLEWVS
SIYPSGGLTYYADSVKG



SLRLSCAASGFTFS


807A-M0046-F05
EVQLLESGGGLVQPGG
FYRMT
WVRQAPGKGLEWVS
SISSSGGGTPYADSVKG



SLRLSCAASGFTFS


807A-M0041-F03
EVQLLESGGGLVQPGG
FYSMF
WVRQAPGKGLEWVS
YIYPSGGWTNYADSVKG



SLRLSCAASGFTFS


807A-M0043-E08
EVQLLESGGGLVQPGG
FYSMG
WVRQAPGKGLEWVS
YIYPSGGGTTYADSVKG



SLRLSCAASGFTFS


807A-M0042-D05
EVQLLESGGGLVQPGG
FYSMV
WVRQAPGKGLEWVS
SISPSGGQTDYADSVKG



SLRLSCAASGFTFS


807A-M0029-G10
EVQLLESGGGLVQPGG
FYTMV
WVRQAPGKGLEWVS
VISPSGGLTHYADSVKG



SLRLSCAASGFTFS


807A-M0046-G03
EVQLLESGGGLVQPGG
FYYMS
WVRQAPGKGLEWVS
RISPSGGLTHYADSVKG



SLRLSCAASGFTFS


807A-M0037-D06
EVQLLESGGGLVQPGG
HYLMV
WVRQAPGKGLEWVS
GISPSGGGTNYADSVKG



SLRLSCAASGFTFS


807A-M0043-E07
EVQLLESGGGLVQPGG
KYPMQ
WVRQAPGKGLEWVS
SISPSGGSTVYADSVKG



SLRLSCAASGFTFS


807A-M0027-E11
EVQLLESGGGLVQPGG
KYSMH
WVRQAPGKGLEWVS
GIYSSGGKTIYADSVKG



SLRLSCAASGFTFS


807A-M0046-A11
EVQLLESGGGLVQPGG
LYGMV
WVRQAPGKGLEWVS
RISPSGGYTGYADSVKG



SLRLSCAASGFTFS


807A-M0041-E01
EVQLLESGGGLVQPGG
LYRMG
WVRQAPGKGLEWVS
SISPSGGWTRYADSVKG



SLRLSCAASGFTFS


807A-M0044-B07
EVQLLESGGGLVQPGG
MYGML
WVRQAPGKGLEWVS
RISPSGGFTNYADSVKG



SLRLSCAASGFTFS


807A-M0028-B02
EVQLLESGGGLVQPGG
MYMMD
WVRQAPGKGLEWVS
SIWPSGGQTWYADSVKG



SLRLSCAASGFTFS


807A-M0039-E06
EVQLLESGGGLVQPGG
NYVMH
WVRQAPGKGLEWVS
VISPSGGATIYADSVKG



SLRLSCAASGFTFS


807A-M0040-A03
EVQLLESGGGLVQPGG
QYNMG
WVRQAPGKGLEWVS
YISSSGGITWYADSVKG



SLRLSCAASGFTFS


807A-M0044-G07
EVQLLESGGGLVQPGG
TYSMH
WVRQAPGKGLEWVS
YIGSSGGFTMYADSVKG



SLRLSCAASGFTFS


807A-M0044-E08
EVQLLESGGGLVQPGG
TYWMI
WVRQAPGKGLEWVS
SISSSGGWTMYADSVKG



SLRLSCAASGFTFS


807A-M0038-A09
EVQLLESGGGLVQPGG
VYSMA
WVRQAPGKGLEWVS
GIWPSGGPTAYADSVKG



SLRLSCAASGFTFS


807A-M0037-C08
EVQLLESGGGLVQPGG
WYAMD
WVRQAPGKGLEWVS
RIRPSGGNTDYADSVKG



SLRLSCAASGFTFS


807A-M0039-H09
EVQLLESGGGLVQPGG
WYAMD
WVRQAPGKGLEWVS
RIRSSGGLTHYADSVKG



SLRLSCAASGFTFS


807A-M0039-D05
EVQLLESGGGLVQPGG
WYAMD
WVRQAPGKGLEWVS
SIYPSGGWTEYADSVKG



SLRLSCAASGFTFS


807A-M0042-F12
EVQLLESGGGLVQPGG
WYAMS
WVRQAPGKGLEWVS
SIYSSGGKTGYADSVKG



SLRLSCAASGFTFS


807A-M0043-H05
EVQLLESGGGLVQPGG
WYAMV
WVRQAPGKGLEWVS
YIRSSGGHTVYADSVKG



SLRLSCAASGFTFS


807A-M0046-H06
EVQLLESGGGLVQPGG
WYDMD
WVRQAPGKGLEWVS
SISSSGGFTTYADSVKG



SLRLSCAASGFTFS


807A-M0042-C03
EVQLLESGGGLVQPGG
WYDMD
WVRQAPGKGLEWVS
SIVSSGGLTDYADSVKG



SLRLSCAASGFTFS


807A-M0040-C03
EVQLLESGGGLVQPGG
WYDME
WVRQAPGKGLEWVS
VIGPSGGPTHYADSVKG



SLRLSCAASGFTFS


807A-M0046-C05
EVQLLESGGGLVQPGG
WYDME
WVRQAPGKGLEWVS
WISSSGGTTWYADSVKG



SLRLSCAASGFTFS


807A-M0027-D05
EVQLLESGGGLVQPGG
WYDMF
WVRQAPGKGLEWVS
SIYSSGITYYADSVKG



SLRLSCAASGFTFS


807A-M0040-B11
EVQLLESGGGLVQPGG
WYDMG
WVRQAPGKGLEWVS
SISPSGGSTTYADSVKG



SLRLSCAASGFTFS


807A-M0039-B02
EVQLLESGGGLVQPGG
WYDMH
WVRQAPGKGLEWVS
SISPSGGLTYYADSVKG



SLRLSCAASGFTFS


807A-M0041-C07
EVQLLESGGGLVQPGG
WYDMH
WVRQAPGKGLEWVS
SISPSGGLTYYADSVKG



SLRLSCAASGFTFS


807A-M0041-H04
EVQLLESGGGLVQPGG
WYDMH
WVRQAPGKGLEWVS
SISSSGGDTTYADSVKG



SLRLSCAASGFTFS


807A-M0028-G07
EVQLLESGGGLVQPGG
WYDMH
WVRQAPGKGLEWVS
YISPSGGWTGYADSVKG



SLRLSCAASGFTFS


807A-M0041-A09
EVQLLESGGGLVQPGG
WYDMN
WVRQAPGKGLEWVS
RISPSGGSTRYADSVKG



SLRLSCAASGFTFS


807A-M0042-B10
EVQLLESGGGLVQPGG
WYDMN
WVRQAPGKGLEWVS
SIGSSGGETRYADSVKG



SLRLSCAASGFTFS


807A-M0041-E06
EVQLLESGGGLVQPGG
WYDMQ
WVRQAPGKGLEWVS
SISSSGGLTTYADSVKG



SLRLSCAASGFTFS


807A-M0037-D10
EVQLLESGGGLVQPGG
WYDMR
WVRQAPGKGLEWVS
SISSSGGRTVYADSVKG



SLRLSCAASGFTFS


807A-M0044-F04
EVQLLESGGGLVQPGG
WYDMS
WVRQAPGKGLEWVS
RIGSSGGATQYADSVKG



SLRLSCAASGFTFS


807A-M0043-D10
EVQLLESGGGLVQPGG
WYDMS
WVRQAPGKGLEWVS
YIVPSGGVTLYADSVKG



SLRLSCAASGFTFS


807A-M0043-G06
EVQLLESGGGLVQPGG
WYDMV
WVRQAPGKGLEWVS
SIVSSGGETRYADSVKG



SLRLSCAASGFTFS


807A-M0037-G01
EVQLLESGGGLVQPGG
WYDMV
WVRQAPGKGLEWVS
YIRPSGGITFYADSVKG



SLRLSCAASGFTFS


807A-M0044-E11
EVQLLESGGGLVQPGG
WYDMV
WVRQAPGKGLEWVS
YIVPSGGETSYADSVKG



SLRLSCAASGFTFS


807A-M0043-A10
EVQLLESGGGLVQPGG
WYDMY
WVRQAPGKGLEWVS
GIGPSGGSTYYADSVKG



SLRLSCAASGFTFS


807A-M0045-B03
EVQLLESGGGLVQPGG
WYDMY
WVRQAPGKGLEWVS
SIRSSGGLTNYADSVKG



SLRLSCAASGFTFS


807A-M0038-A08
EVQLLESGGGLVQPGG
WYDMY
WVRQAPGKGLEWVS
SISPSGGITAYADSVKG



SLRLSCAASGFTFS


807A-M0039-C02
EVQLLESGGGLVQPGG
WYDMY
WVRQAPGKGLEWVS
SISPSGGITAYADSVKG



SLRLSCAASGFTFS


807A-M0027-G01
EVQLLESGGGLVQPGG
WYEMG
WVRQAPGKGLEWVS
SIGPSGGETIYADSVKG



SLRLSCAASGFTFS


807A-M0039-B08
EVQLLESGGGLVQPGG
WYEMG
WVRQAPGKGLEWVS
SIYSSGGQTVYADSVKG



SLRLSCAASGFTFS


807A-M0046-E12
EVQLLESGGGLVQPGG
WYEMN
WVRQAPGKGLEWVS
RIYPSGGPTWYADSVKG



SLRLSCAASGFTFS


807A-M0041-A08
EVQLLESGGGLVQPGG
WYFMH
WVRQAPGKGLEWVS
SIYPSGGTTEYADSVKG



SLRLSCAASGFTFS


807A-M0037-C09
EVQLLESGGGLVQPGG
WYGMG
WVRQAPGKGLEWVS
YISSSGGLTIYADSVKG



SLRLSCAASGFTFS


807A-M0040-G01
EVQLLESGGGLVQPGG
WYGMS
WVRQAPGKGLEWVS
RIVSSGGPTGYADSVKG



SLRLSCAASGFTFS


807A-M0045-E04
EVQLLESGGGLVQPGG
WYGMV
WVRQAPGKGLEWVS
SIYPSGGTTQYADSVKG



SLRLSCAASGFTFS


807A-M0041-H05
EVQLLESGGGLVQPGG
WYNMD
WVRQAPGKGLEWVS
SIGPSGGPTKYADSVKG



SLRLSCAASGFTFS


807A-M0043-A08
EVQLLESGGGLVQPGG
WYNMG
WVRQAPGKGLEWVS
YIGPSGGETYYADSVKG



SLRLSCAASGFTFS


807A-M0038-C09
EVQLLESGGGLVQPGG
WYNMQ
WVRQAPGKGLEWVS
VISPSGGGTYYADSVKG



SLRLSCAASGFTFS


807A-M0042-F09
EVQLLESGGGLVQPGG
WYNMV
WVRQAPGKGLEWVS
WISSSGGMTRYADSVKG



SLRLSCAASGFTFS


807A-M0045-B12
EVQLLESGGGLVQPGG
WYPMY
WVRQAPGKGLEWVS
VIYPSGGHTKYADSVKG



SLRLSCAASGFTFS


807A-M0044-C04
EVQLLESGGGLVQPGG
WYQME
WVRQAPGKGLEWVS
SISSSGGTTDYADSVKG



SLRLSCAASGFTFS


807A-M0026-F11
EVQLLESGGGLVQPGG
WYQMS
WVRQAPGKGLEWVS
SISSSGGHTFYADSVKG



SLRLSCAASGFTFS


807A-M0027-E12
EVQLLESGGGLVQPGG
WYRMT
WVRQAPGKGLEWVS
SISPSGGVTLYADSVKG



SLRLSCAASGFTFS


807A-M0028-B12
EVQLLESGGGLVQPGG
WYSMG
WVRQAPGKGLEWVS
WISSSGGGTPYADSVKG



SLRLSCAASGFTFS


807A-M0026-G08
EVQLLESGGGLVQPGG
WYSMH
WVRQAPGKGLEWVS
SIRSSGGWTKYADSVKG



SLRLSCAASGFTFS


807A-M0043-A07
EVQLLESGGGLVQPGG
WYSML
WVRQAPGKGLEWVS
YIYPSGGATFYADSVKG



SLRLSCAASGFTFS


807A-M0042-F04
EVQLLESGGGLVQPGG
WYSMN
WVRQAPGKGLEWVS
GISSSGGMTHYADSVKG



SLRLSCAASGFTFS


807A-M0045-H09
EVQLLESGGGLVQPGG
WYTMG
WVRQAPGKGLEWVS
SIVSSGGTPYADSVKG



SLRLSCAASGFTFS


807A-M0046-D04
EVQLLESGGGLVQPGG
WYTMH
WVRQAPGKGLEWVS
SIRSSGGMTDYADSVKG



SLRLSCAASGFTFS


807A-M0040-G04
EVQLLESGGGLVQPGG
WYTMQ
WVRQAPGKGLEWVS
SIYPSGGDTKYADSVKG



SLRLSCAASGFTFS


807A-M0045-B01
EVQLLESGGGLVQPGG
WYTMV
WVRQAPGKGLEWVS
SIRSSGGQTKYADSVKG



SLRLSCAASGFTFS


807A-M0040-A08
EVQLLESGGGLVQPGG
WYTMV
WVRQAPGKGLEWVS
SIVPSGGDTHYADSVKG



SLRLSCAASGFTFS


807A-M0026-F05
EVQLLESGGGLVQPGG
YYAMQ
WVRQAPGKGLEWVS
SLYPSGGNTSYADSVKG



SLRLSCAASGFTFS


807A-M0037-H02
EVQLLESGGGLVQPGG
YYGMV
WVRQAPGKGLEWVS
RISPSGGMTDYADSVKG



SLRLSCAASGFTFS


807A-M0042-A06
EVQLLESGGGLVQPGG
YYHMD
WVRQAPGKGLEWVS
SIVSSGGFTAYADSVKG



SLRLSCAASGFTFS


807A-M0028-A07
EVQLLESGGGLVQPGG
YYRMA
WVRQAPGKGLEWVS
SIYSSGGMTLYADSVKG



SLRLSCAASGFTFS


807A-M0043-G01
EVQLLESGGGLVQPGG
YYSMI
WVRQAPGKGLEWVS
RISPSGGQTNYADSVKG



SLRLSCAASGFTFS


807A-M0046-F04
EVQLLESGGGLVQPGG
YYSMT
WVRQAPGKGLEWVS
SISPSGGGTGYADSVKG



SLRLSCAASGFTFS


807A-M0037-F03
EVQLLESGGGLVQPGG
YYSMV
WVRQAPGKGLEWVS
WISSSGGSTNYADSVKG



SLRLSCAASGFTFS


807A-M0039-C10
EVQLLESGGGLVQPGG
YYSMV
WVRQAPGKGLEWVS
WISSSGGSTNYADSVKG



SLRLSCAASGFTFS















Initial Name
FR3
CDR3
FR4







807A-M0027-C11
RFTISRDNSKNTLYLQMNS
AVGYGDYGDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0043-F08
RFTISRDNSKNTLYLQMNS
ELDTAMAPPSDAFDI
WGQGTMVTVSS




LRAEDTAVYYCAR



807A-M0039-E07
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0039-D11
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0037-F10
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0028-B06
RFTISRDNSKNTLYLQMNS
DRGVSLLGAFDI
WGQGTMVTVSS




LRAEDTAVYYCAR



807A-M0046-F05
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0041-F03
RFTISRDNSKNTLYLQMNS
SVVGWGLDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0043-E08
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0042-D05
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0029-G10
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0046-G03
RFTISRDNSKNTLYLQMNS
DFFTSYFDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0037-D06
RFTISRDNSKNTLYLQMNS
GPGYSYGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAK



807A-M0043-E07
RFTISRDNSKNTLYLQMNS
GVTTVPRYYYYYYMDV
WGKGTTVTVSS




LRAEDTAVYYCAR



807A-M0027-E11
RFTISRDNPKNTLYLQMNS
SLDLDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0046-A11
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0041-E01
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0044-B07
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0028-B02
RFTISRDNSKNTLYLQMNS
SVLLDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0039-E06
RFTISRDNSKNTLYLQMNS
DWGPFEAFDI
WGQGTMVTVSS




LRAEDTAVYYCAR



807A-M0040-A03
RFTISRDNSKNTLYLQMNS
DFFTSYFDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0044-G07
RFTISRDNSKNTLYLQMNS
GLYR
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0044-E08
RFTISRDNSKNTLYLQMNS
EDNYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0038-A09
RFTISRDNSKNTLYLQMNS
EDFWSGLEDV
WGKGTTVTVSS




LRAEDTAVYYCAR



807A-M0037-C08
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0039-H09
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0039-D05
RFTISRDNSKNTLYLQMNS
GLGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0042-F12
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0043-H05
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0046-H06
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0042-C03
RFTISRDNSKNTLYLQMNS
QEVWQWPAQFDS
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0040-C03
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0046-C05
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0027-D05
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0040-B11
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0039-B02
RFTISRDNSKNTLYLQMNS
QEVWQWPAQFDS
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0041-C07
RFTISRDNSKNTLYLQMNS
QEVWQWPAQFDS
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0041-H04
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0028-G07
RFTISRDNSKNTLYLQMNS
QEVWQWPAQFDS
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0041-A09
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0042-B10
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LKAEDTAVYYCAR



807A-M0041-E06
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0037-D10
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0044-F04
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0043-D10
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0043-G06
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0037-G01
RFTISRDNSKNTLYLQMNS
QEVWQWPAQFDS
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0044-E11
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0043-A10
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0045-B03
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0038-A08
RFTISRDNSKNTLYLQMNS
QEVWQWPAQFDS
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0039-C02
RFTISRDNSKNTLYLQMNS
QEVWQWPAQFDS
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0027-G01
RFTISRDNSKNTLYLQMNS
DRGVSLLGAFDI
WGQGTMVTVSS




LRAEDTAVYYCAR



807A-M0039-B08
RFTISRDNSKNTLYLQMNS
QEVWQWPAQFDS
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0046-E12
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0041-A08
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0037-C09
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0040-G01
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0045-E04
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0041-H05
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0043-A08
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0038-C09
RFTISRDNSKNTLYLQMNS
VKWDHSPLFDP
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0042-F09
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0045-B12
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0044-C04
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0026-F11
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0027-E12
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0028-B12
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0026-G08
RFTISRDNSKNTLYLQMNS
SSGIYYGYYMDV
WGKGATVTVSS




LRAEDTAVYYCAR



807A-M0043-A07
RFTISRDNSKNTLYLQMNS
GRSTFDI
WGQGTMVTVSS




LRAEDTAVYYCAR



807A-M0042-F04
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTIVTVSS




LRAEDTAVYYCAR



807A-M0045-H09
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0046-D04
RFTISRDNSKNTLYLQMNS
SSGIYYGYYMDV
WGKGATVTVSS




LRAEDTAVYYCAR



807A-M0040-G04
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0045-B01
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0040-A08
RFTISRDNSKNTLYLQMNS
EPIWGYYYYGMDV
WGQGTTVTVSS




LRAEDTAVYYCAR



807A-M0026-F05
RFTISRDNSKNTLYLQMNS
GRGNYDFWSAGYYYY
WGKGTTVTVSS




LRAEDTAVYYCAR
YMDV



807A-M0037-H02
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTPVTVSS




LRAEDTAVYYCAR



807A-M0042-A06
RFTISRDNSKNTLYLQMNS
DRGVSLLGAFDI
WGQGTMVTVSS




LRAEDTAVYYCAR



807A-M0028-A07
RFTISRDNSKNTLYLQMNS
ESIAVAGVDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0043-G01
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0046-F04
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0037-F03
RFTISRDNSKNTLYLQMNS
DLWFGEWDY
WGQGTLVTVSS




LRAEDTAVYYCAR



807A-M0039-C10
RFTISRDNSKNTLYLQMNS
DLWFREWDY
WGQGTLVTVSS




LRAEDTAVYYCAR

















TABLE 11







Amino acid sequences of the VL chains of the antibodies identified using the screening strategies of Examples 21 and 22














Antibody Name
VL-FR1 
VL-CDR1
VL-FR2
VL-CDR2
VI-FR3
VL-CDR3
VL-FR4





807B-M0001-A09
QSVLTQPPSASGTPGQ
SGSSSNIGTYPVN
WYQQLPGAAPKLLIY
STDQRPS
GVPDRFSGSKSGTSASLAISGLQSE
AAWDDSLNGLWV
FGGGTKVTVL



RVTISC



DESDYYC


807B-M0001-B07
QYELTQPPSVSGTPGQ
SGSSSNIGSEYVY
WFQQLPGTAPRLLIY
RNDQRPS
GVPDRFSGSKSGTSASLAISGLQSE
AAWDDSLPGWC
SGGGTKLTVL



RVTISC



DEADYYC


807B-M0001-C10
QYELTQPPSASGTPGQ
SGSSSNIGTNFVY
WYQQLPGTAPKLLIY
RSIKRPS
GVPDRFSGSKSGTSASLAISGLRSE
AAWDDSLSGW
FGGGTKLTVL



TVTISC



DEADYYC


807B-M0001-G03
QSALTQPPSASGTPGQ
SGSSSNIGINSVN
WYQQLPGTAPKLLIY
SNNQRPS
GVPDRFSGSKSGTSASLAISGLRSE
AAWDDSLAGWV
FGGGTKVTVL



RVTFSC



DEADYYC


8076-M0004-A03
QSELTQPPSASGTPGQ
SGSSSNIGSNTVN
WYQQLPGTAPKLLIY
NNNQRPS
GVPDRFSGSKSGTSASLAISGLQSE
AAWHDGLNGPV
FGGGTKLTVL



RVTISC



DEADYYC


807B-M0004-A05
QDIQMTQSPATLSLSP
KASQSVRAFIA
WYQQKPGQAPRLLIS
GASNRAT
GIPDRFSGGGSGTDFTLTISRLEPED
QQYGSSRYT
FGQGTKLEIK



GERATLSC



FAVYYC


8076-M0004-B10
QDIQMTQSPSSLSASV
RASQSISTYLN
WYQQKPGKAPKLLIF
ATSRLQS
EVPSRFSGSGSGTDFTLTISSLQPE
QQSYSPPRT
FGQGTKLDIN



GDRVTITC



DFATYYC


807B-M0004-C01
QDIQMTQSPLSLPVSLG
RSSQSLVHTDGTTYLS
WFQQRPGQSPRRLVY
KVSDRGS
GVPDRFSGSGSGIDFTLKISRVEAE
MQGTHWPPT
FGQGTKLEIK



QKASISC



DVGLYYC


807B-M0004-C04
QDIQMTQSPSSLPVTP
RSSQSLLHSSGYNYLD
WYLQKPGQSPQLLIY
LGSNRAS
GVPDRFTGSGSGTDFTLKISRVEAE
MQALQTPT
FGGGTKVDIK



GEPASISC



DVGVYYC


807B-M0004-C05
QDIQMTQSPATLSVSP
RASQSVSSNLA
WYQQKPGQAPRLLIY
GASTRAT
GVPARFSGSGSGTDFTLSISSLQPE
QQYAGHPIT
FGQGTRLEIK



GERATLSC



DFATYYC


807B-M0004-D10
QSELTQPSSASETPGQ
SGSSSNIGSNLVY
WYQQVPGTALKLLIY
RNDQRPS
GVPDRFSGSKSGTSASLAISGLRSE
VSWDGSLSGWV
FGGGTRLTVL



RVTISC



DEADYFC


807B-M0004-F06
QSELTQAASVSGSPGQ
TGATRDVS
WYQQHPGKAPKLVL
YEVSSRPS
GVSDRFSGSMSGNTASLTISGLQAE
SSTTSRAPRW
FGGGTKLTVL



SITLSC



DEADYYC


807B-M0004-F07
QDIQMTQSPATLSLSP
RASQSVSSYLA
WYQQKPGQAPRLLIY
DAFNRAT
GIPARFSGSGSGTDFTLTISSLEPED
QQRSNWPLT
FGGGTKVEIK



GERATLSC



FAVYYC


807B-M0004-F10
QDIQMTQSPLSLPVTP
RSSQSLMHRNGHHFFD
WYLQKPGQSPQLLIY
WASNRAP
GVPDRFSGSGSGTDFTLKISRVEAE
MQALQTPYT
FGQGTKLEIK



GEPASISC



DVGIYYC


807B-M0004-G08
QDIVMTQTPPSLPVNP
RSSQSLVHSDGNTYLN
WFQQRPGQSPRRLIS
KVSNRDS
GVPDRFSGSGAGTDFTLNISRVEAE
MQVTEFPLT
FGGGTKVEIK



GEPASISC



DVGDYYC


807B-M0004-H03
QDIQMTQSPSSLSASIG
QASQNIDNYLN
WYQQKPGKAPKLLIY
AASSLQS
GVPSRFSGSGSGTDFTLTISSLQPE
QQSYSTPRT
FGQGTKVEIK



DRVTISC



DFATYYC


807B-M0008-A03
QSALTQPASVSGSPGQ
TGTSNDVGGYNYVS
WYQQHPGIAPKVVIY
EVSNRPS
GVSDRFSGSKAGNTASLTISGLQAE
NSYTSSRTWV
FGGGTKVTVL



SITISC



DEADYYC


807B-M0008-A08
QDIQMTQSPSSLSASV
RASQSISSYLN
WYQQKPGKAPKLLIY
AASSLQS
GVPSRFSGSGSGTDFTLTISSLQPE
QQSYSTPLT
FGGGTKVEIK



GDRVTITC



DFATYYC


807B-M0008-B04
QYELTQPPSASGTPGQ
SGTLSNIGTNIVS
WFQQLPGTAPKLLIY
NDHRRPS
GVPDRFSGSKSATSASLAISGLQSE
AAWDDSLNGW
FGGGTKLTVL



RVTISC



DEADYYC


807B-M0008-B08
QDIQMTQSPSSLSASV
RASQSISTYLN
WYQEKPGKAPELLIF
AASSLQG
GVPSRFSGSGSGTDFTLTISSLQPE
QQSYDIPLS
FGGGTKVEIK



GDRVTITC



DLATYYC


807B-M0008-D02
QDIQMTQSPATLSLSLG
RASEYISTYLA
WYQQKPGQAPRLLIY
DASVRAP
GTPARFSGTGSGTDFTLTISSLQPD
QERYDWPLT
FGPGTRLDVK



ERANFSC



DFAVYFC


807B-M0008-D05
QSALTOPPSASGTPGQ
SGSSSNIGRNFVY
WYRQLPGTAPKLLIY
ENNQRPS
GVPDRFSGSKSGTSASLAISGLRSE
AAWDDSLSGLV
FGGGTKLTVL



RVTISC



DEADYHC


807B-M0008-E01
QDIQMTQSPGTLSLSP
RASQSVSSRYLA
WYQQKPGQAPRLLIY
GASSRAP
GIPDRFSGSGSGTDFTLTISSLEPEE
QQYGSSPVT
FGGGTKVEIK



GERGTLSC



SAVYYC


807B-M0008-E06
QYELTQPPSASGTPGQ
SGSSSNIGSNYVY
WHQQLPGTAPKLLIS
RNNQRPS
GVPDRFSGSKSGTSASLAISGLRSE
AAWDDSLSGW
FGAGTKVTVL



RVTISC



DEADYYC


807B-M0008-G11
QSVLTQPASVSGSPGQ
TGASSDVGGSNFVS
WYQQHPGKAPKLIIY
DVSNRPS
GVSNRFSGSKSGNTASLTISGLQAE
SSYTSSSLW
FGGGTKLTVL



SITISC



DDDDTDYYC


807B-M0009-A06
QSVLTQPPSASGTPGQ
SGSSSNIGSYYVY
WYQHLPGTAPKLLIY
RNNQRPS
GVPDRFSGSKSGTSASLAISGLRSE
AAWDDRLSTWV
FGGGTKLTVL



RVSISC



DESDYYC


807B-M0009-A09
QYELTQPPSVSVSPGQ
SGDKLGDKYAS
WYQQKPDQSPVLVIY
QDRKRPS
GIPERFSGSNSGNTATLTISGTQAM
QAWDSNTVV
FGGGTKLTVL



TASITC



DEADYYC


807B-M0009-B07
QDIQMTQSPGTLSLSP
RASQSVSSSYLA
WYQQKPGQAPRLLIY
GASSRAT
GIPDRFSGSGSGTDFTLTISRLEPED
QQYGSSGWT
FGQGTKVEIK



GERATLSC



FAVYYC


807B-M0009-C02
QYELTQPPSVSVSPGQ
SGDKLGDKYTS
WHQQKPGQSPVLVIY
QDRKRPS
GIPERFSGSNSGNTATLTISGTQAM
QAWDSNTVV
FGGGTKLTVL



TASITC



DEADYYC


807B-M0009-C03
QSELTQPPSASGTPGQ
SGSSSNIGSNYVY
WYQQLPGTAPKLLIY
RNNQRPS
GVPDRFSGSKSGTSASLAISGLQSE
AAWDDSLNAWV
FGGGTKLTVL



RVTISC



DEADYYC


807B-M0009-F06
QDIQMTQSPLSLPVTP
KSSQSLLHSNGYNYLD
WYLQKPGQSPQLLIS
LGSNRAS
GVPARFSGSGSGTDFTLKISRVEAE
MQALQTIT
FGQGTRLEIK



GEPASISC



DVGVYYC


807B-M0009-G03
QSVLTQSPSASASLGA
TLSSGHSNYDIA
WHQQQPEKGPRY
LMKLNSD
GIPDRFSGSSSGTERYLTISSLQSED
QTWGTGLRV
FGGGTKLTVL



SVRVTC



EADYYC


807B-M0023-C03
QSELTQPPSVSVSPGQ
SGYDLGAKFVS
WYQQKSGQSPVLVM
YQDNKRPS
GIPERFSGSNSGNTATLTISGTQAM
QVWDSPSYI
FGTGTTVTVL



TATITC



DEADYYC


807B-M0023-G05
QDIQMTQSPGTLSLSP
RASQSVSSSYLA
WYQQKPGQAPRLLIY
GASSRAT
GIPDRFSGSGSGTDFTLTISSLHPED
QQGNSFPIT
FGQGTRLEIK



GERATLSC



FATYFC


807B-M0024-H08
QDIQMTQSPSSLSASV
RASQSISSHLN
WYQQKPGKVPKVLIY
GASRLQS
GVPSRFSGSGSGTDFTLTINSLQPE
QQSYRAPVFT
FGPGTKVDVK



GDRVSITC



DFATYYC


807B-M0025-B05
QSVLTQPPSASGTPGQ
SGSSSNIGRNPVN
WYQHLPGTAPKLLIY
GDNQRPS
GVPDRFSGSRSGTSASLAISGLQSE
AAWDDSLYGPV
FGGGTKLTVL



RVTISC



DEADVYC


807B-M0027-E08
QSALTQPPSVSVSPGQ
AGDELGNKYAS
WYQQKPGQSPVLVIY
QDRKRPS
GIPERFSGSHSGNTATLTISGTQALD
QSWDSSSVI
FGGGTKVTVL



TASITC



EADYYC


807B-M0042-A05
QDIQMTQSPSAMSASV
RASQGISNYLA
WFQQKPGKVPKRLIY
AASSLQS
GVPSRFSGSGSGTEFTLTISSLQPE
LQHNSYPLT
FGGGTKVEIK



GDRVTITC



DFATYYC


807B-M0042-B05
QSALTQPPSASGTPGQ
SGSSSNIGSHYVY
WYQQLPGTDPKLLIY
KSIQRPS
GVPDRFSGSKSGTSASLAISGLRSD
AAWDDSLSGSYV
FGTGTKVTVL



RVTISC



DEGDYYC


807B-M0046-E03
QDIQMTQSPSSVSASV
RASQDISSWLA
WYQQKPGKAPKLLIY
AASRLQS
GVPSRFSGSGSGTDFSLTISSLQPD
QQSHSFPLS
FGGGTKVEIK



GDRVTLTC



DFATYYC


807B-M0050-A04
QDIQMTQSPSSLSVSP
RASQTISNDLA
WYQQTPGQAPRLLIY
GASSRAT
GIPDRFSGSGSGTDFTLTISNLQPED
QQADSFPLT
FGGGTKVEIK



GERATLSC



FATYFC


807B-M0050-B09
QDIQMTOSPDSLAVSL
QSSQSVLYSSNNKNYLA
WYQQKPGQPPKLLIY
GASTRES
GVPDRFSGSGSGTDFTLTISSLQAE
QQYYTTPLT
FGGGTKVEIK



GERATINC



DVAVYYC


807B-M0050-E04
QDIQMTQSPSSVSASV
RASQGISSWLA
WYQQKPGKAPKLLIY
PASSLQS
GVPSRFSGSGSGTDFTLTISSLQPE
QQGTSFPLT
FGGGTKVEIK



GDRVTITC



DFATYYC


807B-M0050-G01
QDIQMTQSPSTLSASV
RASQSISTWLA
WYQQKPGKAPKLLIY
KAFSLEG
GVPSRFSGSGSGTEFTLTISSLQPE
QQSYSPPLT
FGGGTKVDIR



GDRVTITC



DFATYYC


807B-M0050-G05
QDIQMTQSPSSLSASV
RASOGISNYLA
WYQQKPGKVPKLLIY
AASTLQS
GVPSRFSGSGSGTDFTLTISSLQPE
QNYNRAPRT
FGQGTKVEIK



GDRVTITC



DVATYYC


807B-M0050-H05
QDIQMTQSPPSVSASV
RASQVITRWLA
WYQQKPGQAPKLLIY
SASSLQS
GVPSRFSGSGSGTDFTLTISSLQPE
QQATSFPLT
FGGGTKVEIK



GDRVTITC



DFATYYC


807B-M0050-H10
QDIQMTQSPLSLSASV
RASQSISSYLN
WYQHKPGKAPRLLIY
GASSLQN
GVPSRFTGSGTGTDFTLTISSLQPE
QQSFTTPLT
FGGGTKVEIK



GDRVTITC



DFATYYC
















TABLE 12







Amino acid sequences of the VH chains of the antibodies identified using the screening strategies of Examples 21 and 22














Isolate Name
VH-FR1
VH-CDR1
VH-FR2
VH-CDR2
VH-FR3
VH-CDR3
VH-FR4





807B-M0001-A09
EVQLLESGGGLVQPGGSLR
RYPMF
WVRQAPGKGLEW
SISSSGGYTVYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
VGKGAYYYAMDV
WGQGTTVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0001-B07
EVQLLESGGGLVQPGGSLR
FYGMV
WVRQAPGKGLEW
SISPSGGYTLYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
DGRRPHYGSGRWAY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAK


807B-M0001-C10
EVQLLESGGGLVQPGGSLR
QYVMF
WVRQAPGKGLEW
SISSSGGKTSYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
RLKIYDSSGYYYYYGMDV
WGQGTTVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0001-G03
EVQLLESGGGLVQPGGSLR
SYAMS
WVRQAPGKGLEW
SISPSGGFTPYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
VGKGAYYYAMDV
WGQGTTVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0004-A03
EVQLLESGGGLVQPGGSLR
RYLMM
WVRQAPGKGLEW
VISPSGGRTWYADSV
RFTISRDNSKNTLYLQMNSLRAE
SIAAAGTDY
WGQGTLVTVSS



LSCAASGFTFS

VS
KG
DTAVYYCVR


807B-M0004-A05
EVQLLESGGGLVQPGGSLR
NYFMI
WVRQAPGKGLEW
WISPSGGTTQYADSV
RFTISRDNSKNTLYLQMNSLRAE
EAGY
WGQGTLVTVSS



LSCAASGFTFS

VS
KG
DTAVYYCAR


807B-M0004-B10
EVQLLESGGGLVQPGGSLR
KYVMI
WVRQAPGKGLEW
SISPSGGDTTYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
DLGSNWGTGVVWN
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0004-C01
EVQLLESGGGLVQPGGSLR
NYNMH
WVRQAPGKGLEW
SIYSSGGTTLYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
LVADEWIDAFDI
WGQGTMVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0004-C04
EVQLLESGGGLVQPGGSLR
AYYMG
WVRQAPGKGLEW
VIRPSGGKTKYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
GPHGQGGVDS
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0004-C05
EVQLLESGGGLVQPGGSLR
EYFMT
WVRQAPGKGLEW
SIRPSGGKTRYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
VSRYYNNGAYRLDAFDI
WGPGTVVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0004-D10
EVQLLESGGGLVQPGGSLR
WYQMS
WVRQAPGKGLEW
VISPSGGRTIYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
QGLLTAFDI
WGQGTMVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0004-F06
EVQLLESGGGLVQPGGSLR
AYRMA
WVRQAPGKGLEW
YISSSGGVTSYADSVKG
RFTISRDNSKNTLYLQMKSLRAE
GTHLPGVDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0004-F07
EVQLLESGGGLVQPGGSLR
EYYMT
WVRQAPGKGLEW
SIRPSGGATRYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
EPIWGYYYYGMDV
WGQGTTVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0004-F10
EVQLLESGGGLVQPGGSLR
GYIMA
WVRQAPGKGLEW
GIGSSGGLTAYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
EAGY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0004-G08
EVQLLESGGGLVQPGGSLR
TYAMA
WVRQAPGKGLEW
SIRSSGGVTKYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
GGAVAGY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0004-H03
EVQLLESGGGLVQPGGSLR
SYPMV
WVRQAPGKGLEW
GIWSSGGLTYYADSV
RFTISRDNSKNTLYLQMNSLRAE
EGSAGVVKGPARYYYYY
WGKGTTVTVSS



LSCAASGFTFS

VS
KG
DTAVYYCAR
MDV


807B-M0008-A03
EVQLLESGGGLVQPGGSLR
HYTMY
WVRQAPGKGLEW
GISPSGGVTPYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
AGSGGSFDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0008-A08
EVQLLESGGGLVQPGGSLR
KYLMM
WVRQAPGKGLEW
YIWPSGGTDYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
VRTSRINGSGFDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAK


807B-M0008-B04
EVQLLESGGGLVQPGGSLR
RYPMS
WVRQAPGKGLEW
SISPSGGPTSYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
SGPYYFDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0008-B08
EVQLLESGGGLVQPGGSLR
HYPMS
WVRQAPGKGLEW
SISPSGGFTMYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
FEYSSSSGISWFDP
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0008-D02
EVQLLESGGGLVQPGGSLR
KYGMT
WVRQAPGKGLEW
SIRPSGGITKYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
ENYGPDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0008-D05
EVQLLESGGGLVQPGGSLR
TYHMF
WVRQAPGKGLEW
GISSSGGSTRYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
VSVTTNAFDI
WGQGTMVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0008-E01
EVQLLESGGGLVQPGGSLR
TYAMT
WVRQAPGKGLEW
SISSSGGGTKYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
HGYSSGWPPFDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0008-E06
EVQLLESGGGLVQPGGSLR
HYPMS
WVRQAPGKGLEW
SIVPSGGYTLYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
HNRAIGTFDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0008-G11
EVQLLESGGGLVQPGGSLR
SYAMI
WVRQAPGKGLEW
GISPSGGQTVYADSV
RFTISRDNSKNTLYLQMNSLRAE
AGSGGSFDY
WGQGTLVTVSS



LSCAASGFTFS

VS
KG
DTAVYYCAR


807B-M0009-A06
EVQLLESGGGLVQPGGSLR
VYNMV
WVRQAPGKGLEW
VISPSGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
DRGYCSGNTCYIDAFDI
WGQGTMVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0009-A09
EVQLLESGGGLVQPGGSLR
RYPMA
WVRQAPGKGLEW
GISSSGGLTSYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
FVGAKPADY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0009-B07
EVQLLESGGGLVQPGGSLR
FYWMV
WVRQAPGKGLEW
GISPSGGPTKYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
EPIWGYYYYGMDV
WGQGTTVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0009-C02
EVQLLESGGGLVQPGGSLR
SYPMT
WVRQAPGKGLEW
GISSSGGSTAYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
VTGGDFDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAK


807B-M0009-C03
EVQLLESGGGLVQPGGSLR
KYQMT
WVRQAPGKGLEW
VISSSGGDTAYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
DRGYCSGNTCYIDAFDI
WGQGTMVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0009-F06
EVQLLESGGGLVQPGGSLR
PYWMF
WVRQAPGKGLEW
GIVSSGGMTGYADSV
RFTISRDNSKNTLYLQMNSLRAE
VGMSTYAFDI
WGQGTMVTVSS



LSCAASGFTFS

VS
KG
DTAVYYCAR


807B-M0009-G03
EVQLLESGGGLVQPGGSLR
PYRMA
WVRQAPGKGLEW
SISPSGGHTGYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
ESDGTTSAYFDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0023-C03
EVQLLESGGGLVQPGGSLR
SYMMG
WVRQAPGKGLEW
YIYPSGGWTYYADSV
RFTISRDNSKNTLYLQMNSLRAE
GRSWGRYFQH
WGQGTLVTVSS



LSCAASGFTFS

VS
KG
DTAVYYCAR


807B-M0023-G05
EVQLLESGGGLVQPGGSLR
MYWMG
WVRQAPGKGLEW
SISPSGGFTMYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
GLYR
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0024-H08
EVQLLESGGGLVQPGGSLR
AYNMD
WVRQAPGKGLEW
SIYPSGGHTNYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
GKRIAARGGYYFDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0025-B05
EVQLLESGGGLVQPGGSLR
NYSMV
WVRQAPGKGLEW
SIVPSGGFTLYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
HGSSWTFDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0027-E08
EVQLLESGGGLVQPGGSLR
NYRME
WVRQAPGKGLEW
SIWSSGGLTKqADSVKG
RFTISRDNSKNTLYLQMNSLRAE
GLYR
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0042-A05
EVQLLESGGGLVQPGGSLR
HYQMK
WVRQAPGKGLEW
SIGSSGGSTSYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
GPL
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0042-B05
EVQLLESGGGLVQPGGSLR
NYHMD
WVRQAPGKGLEW
SISPSGGITKYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
GVGATAGI
WGQGTMVTVSS



LSCAASGFTFS

VS

DTSVYYCAG


807B-M0046-E03
EVQLLESGGGLVQPGGSLR
PYAMI
WVRQAPGKGLEW
YISPSGGKTQYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
DQQTDYFDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAK


807B-M0050-A04
EVQLLESGGGLVQPGGSLR
QYNMN
WVRQAPGKGLEW
GISSSGGPTVYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
FRCTSTSCFSDY
WGQGTLVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0050-B09
EVQLLESGGGLVQPGGSLR
NYGML
WVRQAPGKGLEW
VISSSGGYTFYADSVKG
RFTISRDNSKNSLYLQMNSLRAE
VGATGPFDI
WGQGTMVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0050-E04
EVQLLESGGGLVQPGGSLR
RYSMM
WVRQAPGKGLEW
GISPSGGPTSYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
ENIEYSSSFPNMGRPHYY
WGQGTTVTVSS



LSCAASGFTFS

VS

DTAVYYCAR
YYYGMDV


807B-M0050-G01
EVQLLESGGGLVQPGGSLR
RYEMY
WVRQAPGKGLEW
VISSSGGTTFYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
GGDNWNYLSV
WGQGTTVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0050-G05
EVQLLESGGGLVQPGGSLR
PYPMD
WVRQAPGKGLEW
VISSSGGTTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
VGMSTYAFDI
WGQGTMVTVSS



LSCAASGFTFS

VS

DTAVYYCAR


807B-M0050-H05
EVQLLESGGGLVQPGGSLR
NYVMH
WVRQAPGKGLEW
SIGPSGGGTEYADSV
RFTISRDNTKNTLYLQMNSLRAE
DRGYCSGNTCYIDAFDI
WGQGTMVTVSS



LSCAASGFTFS

VS
KG
DTAVYYCAR


807B-M0050-H10
EVQLLESGGGLVQPGGSLR
TYPMQ
WVRQAPGKGLEW
VISSSGGYTQYADSVKG
RFTISRDNSKNTLYLQMNSLRAE
MRVDYGDNYGMDV
WGQGTTVTVSS



LSCAASGFTFS

VS

DTAVYYCAS




















TABLE 13









Enrichment
Peptide Mapping
























Isolate Name
Peptide
VH
CDR3
1
2
3
4
5
6
7
8
9
10
bCTD
Fab IHC
Group


























807B-MO001-A09
E22-P1
6
8
+














807B-MO001-B07
E22-P1
2
2
+




+




+
+/−
4


807B-MO001-C10
E22-P1
1
1
+


807B-MO001-G03
E22-P1
2
8
+


807B-MO042-A05
E22-P2
1
1

+


807B-MO042-B05
E22-P2
7
7

+


807B-MO050-A04
E22-P3
19
19


+


807B-MO050-B07
E22-P3
20
20


+







+


807B-MO050-B09
E22-P3
4
4


+


807B-MO050-E04
E22-P3
1
1


+


807B-MO050-G01
E22-P3
2
2


+


807B-MO050-G05
E22-P3
2
3


+


807B-MO050-H05
E22-P3
1
8


+


807B-MO050-H10
E22-P3
1
1


+


807B-MO004-A03
E22-P4
5
5



+




+

+
+
5


807B-MO004-A05
E22-P4
2
3



+







+/−
1


807B-MO004-B10
E22-P4
3
3



+


807B-MO004-C01
E22-P4
2
2



+


807B-MO004-C04
E22-P4
1
1



+







+/−
1


807B-MO004-C05
E22-P4
1
1



+







+/−
1


807B-MO004-D10
E22-P4
1
1



+


807B-MO004-F06
E22-P4
2
2



+







+/−
1


807B-MO004-F07
E22-P4
1
4



+


807B-MO004-F10
E22-P4
1
3



+




+


+/−
2


807B-MO004-G08
E22-P4
1
1



+


807B-MO004-H03
E22-P4
1
1



+




+


+
2


807B-MO023-C03
E23-P4
1
1



+


807B-MO023-G05
E23-P4
1
149



+


807B-MO024-H08
E23-P4
4
4



+


807B-MO046-E03
E22-P7
3
3






+


807B-MO008-A03
E22-P8
2
3







+


807B-MO008-A08
E22-P8
1
1







+


807B-MO008-B04
E22-P8
2
2







+


807B-MO008-B08
E22-P8
1
1







+


+


807B-MO008-D02
E22-P8
5
5







+


807B-MO008-D05
E22-P8
2
2







+


807B-MO008-E01
E22-P8
2
2







+


807B-MO008-E06
E22-P8
1
1







+


807B-MO008-G11
E22-P8
1
3







+


807B-MO008-G12
E22-P8
1
1







+


807B-MO025-B05
E23-P8
1
1







+


+


807B-MO027-E08
E23-P8
148
149



+



+


+
+
6


807B-MO009-A06
E22-P9
5
8



+




+


807B-MO009-A09
E22-P9
15
15








+


807B-MO009-B07
E22-P9
4
5
+
+
+/−
+
+
+
+
+
+
+
+


807B-MO009-C02
E22-P9
13
13








+


807B-MO009-C03
E22-P9
2
8



+




+

+
+/−
5


807B-MO009-F06
E22-P9
1
3








+


+/−
3


807B-MO009-G03
E22-P9
1
1



+




+
















TABLE 14





Amino acid sequences of the VL chains of the antibodies identified using the screening strategy of Example 23



















Isolate Name
FR1
CDR1
FR2
CDR2





807B-M0011-C07
QYELTQPPSASGSP
TGTSSDVGTYKYVS
WYQQHPDKAPRLIIY
EVNRRPS



GQSVTISC


807B-M0012-C09
QSELTQPPSASGTP
SGTLSNIGTNIVS
WFQQLPGTAPKLLIY
NDHRRPS



GQRVTISC


807B-M0012-D09
QDIQMTQSPATLSLS
RASQSVSSYLA
WYQQKPGQAPRLLIY
DASNRAT



PGERATLSC


807B-M0012-F10
QDIQMTQSPSFLSA
RASQGISNYLA
WYQQKPGKAPKLLIY
VASALQS



SVGDRVTITC


807B-M0012-F12
QDIQMTQSPATLSV
RASQSVSSNLA
WYQQKPGQAPRLLIY
AASSLQS



SPGERATLSC


807B-M0012-G05
QDIQMTQSPGTLSL
RASRSLFSTYLA
WYQQKPGQPPRLLIY
GASTRAT



SPGERATLSC


807B-M0013-A12
QDIQMTQSPSTLSA
RASQSISRWLA
WYQQKPGKAPKLLIY
AASSLQS



SVGDRVTITC


807B-M0013-B04
QDIQMTQSPSSLPA
RASQNIVTYLN
WYQQKPGKAPKLLIY
AASSLQS



SVGDRVTITC


807B-M0013-C03
QDIQMTQSPDSLSA
RASQSISSYLN
WYQQKPGKAPKLLIY
AASSLQS



SVGDRVTITC


807B-M0013-F06
QYELTQPPSASGTP
SGSGSNIGSNLVY
WYQQLPGTAPKLLIY
RNTQRPS



GQRVTISC


807B-M0013-G05
QDIQMTQSPGTLSL
RASQSLSSSYLA
WYQQKPGQPPRLLIY
GASRRAT



SPGERATLSC


807B-M0014-D07
QDIQMTQSPATLSLS
RASQSVSRYVA
WYQQKPGQSPRLVIY
DASNRAT



PGESTTLSC


807B-M0014-D09
QDIQMTQSPSSFSA
RASQGVGSYLA
WYQQKPGKAPKLLIY
GAYTLQS



STGDRVTITC


807B-M0014-E08
QDIQMTQSPSSLSA
RASQDIRDDLG
WYQQKPGKAPKRLIY
AASSLQS



SVGDRVTITC


807B-M0014-F07
QDIQMTQSPSSLSA
RASQSIDTYLN
WYQQKPGKAPKLLIY
AASKLED



SVGDRVAITC


807B-M0016-C06
QDIQMTQSPSSLSA
RASQSINTYLN
WYQQKPGQAPKLLIY
ASSTLQR



SVGDRVTITC


807B-M0016-D01
QDIQMTQSPSSLSA
RASQSIDNYLN
WYQQKPGIAPKLLIY
TASSLQS



SVGDRVTITC


807B-M0016-D08
QDIQMTQSPSSLSA
RASQSISSFLN
WYQQKPGKAPNLLIY
GASSLQS



SVGDRVAITC


807B-M0016-E01
QDIQMTQSPSSLSA
RASQSIGRYLN
WYQQKPGKAPKLLIY
TASSLQS



SVGDRITITC


807B-M0016-F04
QDIQMTQSPATLSA
RASQSVNNWVA
WYQQKPGGAPEGLIY
KASHLQS



SVGDRVTFTC


807B-M0016-F05
QDIQMTQSPSSLSA
RASQSISTYLN
WYQQKPGKAPKLLIS
APSRLQS



SVGDSVTITC


807B-M0016-F08
QSALTQPRSVSGSP
TGTSSDVGGYNYVS
WYQQHPGKAPKLMIY
DVSKRPS



GQSVTISC


807B-M0017-B05
QDIQMTQSPGTLSL
RASQSVHSSYLA
WYQQKPGQAPRLLIY
GTSSRAT



SPGERATLSC


807B-M0017-B06
QYELTQPPSASGSP
TGTSSDVGAYNYVS
WYKIHPGKAPKLMIY
DVNNRPS



GQSVTISC


807B-M0017-E05
QDIQMTQSPGTLSL
RASQSVTSSYLA
WYQQKPGQAPRLLIY
GASSRAT



SPGERATLSC


807B-M0018-C12
QSELTQPPSASGSP
TGSSRDIGNYNYVS
WYQQFPGKAPKLIIY
DVRKRPS



GQTVTISC


807B-M0018-E09
QDIQMTQSPGTLSL
RASQSVSSSYLA
WYQQKPGQAPRLLIY
GASSRAT



SPGERATLSC


807B-M0018-G02
QDIQMTQSPSSLSA
RASQDIRDDLG
WYQQKPGKAPKRLIY
AASSLQS



SIGDRVTITC


807B-M0019-A04
QDIQMTQSPGTLSL
RASQSVSSSYLA
WYQQKPGQAPRLLIY
GASSRAT



SPGERATLSC


807B-M0019-A10
QDIQMTQSPLSLSAS
RASQDIRSDLG
WYQQKPGKAPKLLIY
GASTLQS



IGDRVTITC


807B-M0019-C01
QSALTQPASVSGSP
SGTSSDVGGYNYVS
WYQQHPGKAPKLVIY
DVSNRPS



GQPITISC


807B-M0019-F06
QSELTQPASVSGSP
TGTSSDVGSYNLVS
WYQQHPGKAPKLMIY
EGSKRPS



GQSITISC


807B-M0019-G07
QDIQMTQSPATLSA
RASQGLASWLA
WYQQKPGKAPNLLIY
KASNLKS



SVGDRVTITC


807B-M0020-D01
QDIQMTQSPSSLSA
RASQTIRDYLH
WYQQKPGKAPKLLIY
AASSLQV



SVGDRVSIT.


807B-M0020-F06
QDIQMTQSPGTLSL
RASQSISYNYLA
WYQQKPGQAPRLLIY
DASNRAT



SPGERATLSC


807B-M0020-F12
QDIQMTQSPSSLSA
RASQDIRNYLA
WFQQKPGKAPKSLIY
GASSLQG



SVGDRVTITC


807B-M0020-G01
QSELTQPASVSGSP
TGTSSDVGSYNLVS
WYQQHPGKAPKLMIY
EGSKRPS



GQSITISC


807B-M0079-B09
QDIQMTQSPSSLSA
RASQSISSYLN
WYQQKPGNAPRLLIY
SASTLNS



SVGDRVTITC


807B-M0079-D10
QSALTQPPSVSVSP
AGDELGNKYAS
WYQQKPGQSPVLVIY
QDRKRPS



GQTASITC


807B-M0079-H01
QDIQMTQSPSSLSA
RASQSISSYLN
WYQQKPGKAPKLLIY
AASSLQS



SVGDRVTITC


807B-M0079-H05
QDIQMTQSPSSLSA
RASQGINSWLA
WYQQRPGKAPRSLIY
AATNLQN



SVGDRVTITC


807B-M0080-A02
QDIQMTQSPLSLSAS
RASQSISTYLN
WYQQKPGKAPKILIY
AASSLQS



VGDRVTIPC


807B-M0080-C04
QSALTQPPSASGTP
SGSTSNIGSNNVN
WYQQLPGTAPKLLMY
TTNYRPS



GQTVAISC


807B-M0080-F10
QDIQMTqSPSSVSAS
RASQGISIWLA
WYQQKPGKAPKLLIY
GASSLQS



IGDRVTITC


807B-M0081-C03
QDIQMTQSPSTLSA
RASQSINRWLA
WYQQKPGKAPKLLIY
KASNLES



SVGDRVTITC


807B-M0081-C05
QSALTQPPSVSGAP
TGSSSNIGAPYDVH
WYQQVPGTAPKVLIY
GNNHRPS



GQRVTISC


807B-M0081-D08
QDIQMTQSPSSVSA
RASQGISSWLA
WYQQKPGKAPKLLIY
AASSLQS



SVGDRVTITC


807B-M0081-E08
QDIQMTQSPSSLPA
RASRNIGKYLN
WYQQIRGRAPRLLVY
LASSVQT



SVGDRVTITC


807B-M0081-F12
QDIQMTqSPSSLSAS
RASQSISSYLN
WYQQKPGKAPKLLIY
AASSLQS



VGDRVTITC


807B-M0081-G04
QDIQMTQSPDTLSLS
RASQSISTSLA
WYHQRPGQAPRLLIY
DASNRAT



PGERATLSC


807B-M0081-G11
QDIQMTQSPSSLSA
RASQSIDTYLN
WYQQKPGKAPKLLIY
AASKLED



SVGDRVAITC


807B-M0081-H03
QYELTQPPSASGSP
TGTSSDVGGYNYVS
WYQQHPGKAPKFMIY
EVNKRPS



GQSVTISC


807B-M0081-H07
QDIQMTQSPSSLSA
RASQSISSYLN
WYQQKPGKAPKLLIY
AASSLQS



SVGDRVTITC


807B-M0082-B07
QSALTQPASVSGSP
TGTSSDVGGYNYVS
WYQQHPGKAPKLMIY
DVSNRPS



GQSITISC


807B-M0082-E01
QDIQMTQSPSSLSA
RASQSIDTYLN
WYQQKPGKAPKLLIY
AASSLQS



SVGDRVAITC


807B-M0082-E08
QDIQMTQSPSSLSA
RASQSISSYLN
WYQQKQGKAPKLLMF
AASSLKS



SIGDRVTITC


807B-M0082-H06
QDIQMTQSPSSLSA
RASQGIRNNLA
WYQQRPGKAPKRLIY
GASNLHS



SVGDRVTVTC


807B-M0083-B10
QDIQMTQSPSSVSA
RASQDIHTWLA
WYQQKPGKAPKLLIY
AASSLQS



SVGDRVTIIC


807B-M0083-E10
QDIQMTQSPGTLSL
RASQSISSRYLA
WYQQKAGQAPRLLMY
GASRAT



SPGERATLSC


807B-M0083-E11
QSALTQPPSVSVAP
GGNNIGTKIVN
WYQQRPGQAPVVVVY
DNSDRPS



GQTARITC


807B-M0084-C03
QYELTQPPSVSVAP
GGSNIGSKSVH
WYQQKSGQAPVLVVY
DDSDRPS



GQTARISC


807B-M0084-C11
QDIQMTQSPSSLSA
RASQSIATFLN
WYQQKPGKAPNLLIS
GAFNLQS



SVGDRVTITC


807B-M0084-E07
QDIQMTQSPSSLPA
RASQSISRYLN
WYQQKPGKAPKVMIY
DASTLQS



SVGDRVTITC


807B-M0084-F03
QDIQMTQSPATLSV
RASQSVSNNLA
WYQQKPGQAPRLLIY
AASTRAT



SPGERATLSC


807B-M0084-F08
QDIQMTQSPATLSV
RASQSVRNNLA
WYQQKPGQAPRLLIY
GASTRAT



SPGARATLSC


807B-M0084-H05
QSALTQPPSVSAAP
SGGTSNIQYNGVN
WYQQLPGKAPKLLIY
FDDLLPS



GQRVTISC


807B-M0085-B12
QDIQMTQSPSSVSA
RASQGISSWLA
WYQQKPGKAPKLLIY
AASSLQS



SVGDRVTITC


807B-M0085-C01
QDIQMTQSPATLSV
RASQSVSSNLA
WYQQKPGQAPRLLIY
GASTRAT



SPGERATLSC


807B-M0085-E10
QDIQMTQSPSSLSA
QASQDISKYLN
WYQQRPGKAPELLIY
DASNLEP



SVGDRVTITC


807B-M0085-G03
QDIQMTQSPLSLSAS
RASQSISSYLN
WYQQKPGKAPKLLIY
AASSLQS



VGDRVTITC


807B-M0085-G07
QDIQMTQSPGTLSL
RASQSVSSSYLA
WYQQKPGQAPRLLIY
GASSRAT



SPGERATLSC


807B-M0085-G08
QDIQMTQSPGTLSL
RASQSVSRSSLA
WYQQKPGQAPRLLIY
GASSRAT



SPGERATLSC


807B-M0086-C06
QSALTQPPSASGTP
SGGSSNIGSNIVN
WYQQVPGMAPKLLYT
TNNRRPS



GQKVTISC

NNRRPS


807B-M0086-D03
QDIQMTQSPSSLSA
RASQSISSYLN
WYQQKPGKAPKLLIY
VASSLQS



SVGDRVTITC


807B-M0086-E08
QDIQMTQSPGTLSL
SVSQSVSSNYLA
WYQQKPGQSPRLLIY
GASARAT



SPGERATLSC


807B-M0086-G03
QDIQMTqSPSFLSAS
RASQVLRRPLA
WYQQKAGKAPKLLIS
AFSILES



VGDRVSVTC















Isolate Name
FR3
CDR3
FR4







807B-M0011-C07
GVPDRFSGSKSGNTASLTI
YSHATGNNYV
FGTGTKVTVL




SGLQAEDEADYYC



807B-M0012-C09
GVPDRFSGSKSATSASLAI
AAWDDSLNGVV
FGGGTKLTVL




SGLQSEDEADYYC



807B-M0012-D09
GIPARFSGSGSGTDFTLTI
QQRSNWPRYT
FGQGTKLEIK




SSLEPEDFAVYYC



807B-M0012-F10
GVPSRFSGSGSGTEFTLTI
QQYYSYSA
FGQGTRVEIK




SSLQPEDFATYYC



807B-M0012-F12
GVPSRFSGSGSGTDFTLT
QQTYSTPWT
FGQGTKLEIK




VSSLQPEDFATYYC



807B-M0012-G05
GIPDRFSGSGSGTDFTLTI
QQYVSSQLT
FGGGTKVEIK




SRLEPEDSALYYC



807B-M0013-A12
GVPSRFSGSGSGTDFTLTI
QQSYSTPLT
FGGGTKVEIK




SSLQPEDFATYYC



807B-M0013-B04
GVPSRFSGSGSGTDFTLTI
QQSYSMSSWT
FGQGTNLEIK




SSLQPEDFATYYC



807B-M0013-C03
GVPSRFSGSGSGTDFTLTI
QQSYSTPPYT
FGQGTKLEIK




SSLQPEDFATYYC



807B-M0013-F06
GVPDRFSASKSGTSASLAI
ATWDDSLGGVV
FGGGTKLTVL




SGLRSEDEADYHC



807B-M0013-G05
GIPDRFSGSGSGTDFTLTI
QHYGRSPLFT
FGPGTTVDIK




SRLEPEDFAVYYC



807B-M0014-D07
GIPARFSGSGSGTDFTLTIT
LQRSNWPFT
FGPGTKVEIK




SLEPEDFGIYYC



807B-M0014-D09
GVPSRFSGSGSGTDFTLII
QQYYSYPFT
FGPGTKVDIK




SGLQSEDFATYYC



807B-M0014-E08
GVPSRFSGSGSGTEFTLTI
QQHNNYPSFT
FGPGTRLDIK




SSLQPEDFATYYC



807B-M0014-F07
GVPSRFSGSGTGTDFTLTI
QQSYSSPGIT
FGPGTKVEIK




RSLQPEDFASYFC



807B-M0016-C06
GVPSRFSGSGSGTDFTLTI
QQSYSPPLYT
FGQGTKLDLK




SSLQLEDFATYFC



807B-M0016-D01
GVPSRFSGSGSGTDFTLTI
QQSYTTPHT
FGQGTKLEIR




SSLQPEDFATYYC



807B-M0016-D08
GVPSRFSGSGSGTDFTLTI
QQSYSTPYT
FGQGTKLEIK




SSLQPEDFATYYC



807B-M0016-E01
GVPSRFSGSGSGTDFTLTI
QQSFTTPHT
FGLGTKLEIE




SSLQPEDFATYYC



807B-M0016-F04
GVPSRFSGGGSGTVFTLTI
QQYQTYPYT
FGQGTRLDMK




TSLQPDDFATYYC



807B-M0016-F05
GVPSRFSDSGSGTDFTLAI
QQSYSTPVT
FGQGTKLEIK




SSLQPEDFATYYC



807B-M0016-F08
GVPDRFSGSKSGNTASLTI
CSYAGNYSVV
FGGGAKLSVL




SGLQAEDEADYYC



807B-M0017-B05
GIPDRFSGNGFGTDFTLTI
QQYGSSPIT
FGQGTRLEIK




SRLEPEDFAVYYC



807B-M0017-B06
GVSNRFSGSKSGNTASLTI
CSYAGSSTQV
FGTGTKVTVL




SGLQAEDEADYYC



807B-M0017-E05
GIPDRFSGSGSGADFTLTI
QQYGTSPYT
FGQGTKLEIK




SRLEPEDFAVYYC



807B-M0018-C12
GVSDRFSGSKSGNTAFLT
GSYTGTNNV
FGPGTSVTVL




VSGLQTEDEADYFC



807B-M0018-E09
GIPDRFSGSGSGTDFTLTI
QQYGSSRVT
FGGGTKVEIK




SRLEPEDFAVYYC



807B-M0018-G02
GVPSRFSGSGSGTEFTLTI
LQHNTFPSFT
FGPGTKVDIK




SSLQPEDFATYYC



807B-M0019-A04
GIPDRFSGSGSGTDFTLTI
QQYGSSSIT
FGQGTRLEIK




SRLEPEDFAVYYC



807B-M0019-A10
GVPSRFSGSGSGADFTLII
LQDYNYPRT
FGQGTKVEIK




SNLQPEDFATYYC



807B-M0019-C01
GISYRFSGSKSVNTASLTIS
SSYTSNSTLV
FGGGTQADRP




GLQAEDEADYFC



807B-M0019-F06
GVSNRFSGSKSGNTASLTI
CSYAGSSTLV
FGGGTKLTVL




SGLQAEDEADYYC



807B-M0019-G07
GVPSRFSGSESGTEFTLTI
HQYYSNSWT
FGQGTKVEIK




SSLQPDDFATYFC



807B-M0020-D01
GVPSRFSGSGSGTDFTLTI
QQTYSTLIT
FGQGTRLEIK




SSLQPEDLATYYC



807B-M0020-F06
GIPARFSGSGSGTDFTLTI
QQRSNWPPGLT
FGGGTKVEIK




SSLEPEDFAVYYC



807B-M0020-F12
GVPSKFSGSGSGTDFTLTI
QQYNSYPLT
FGGGTKVEVK




SGLQPEDFATYYC



807B-M0020-G01
GVSNRFSGSKSGNTASLTI
CSYAGSSTYV
FGTGTKVTVL




SGLQAEDEADYYC



807B-M0079-B09
GVPSRFSGSGSGTHFTLTI
QQANSLPFT
FGQGTKLEIK




SSLQPEDFGIYYC



807B-M0079-D10
GIPERFSGSHSGNTATLTI
QSWDSSSVI
FGGGTKVTVL




SGTQALDEADYYC



807B-M0079-H01
GVPSRFSGSGSGTDFTLTI
QQSYSTPFT
FGPGTKVDIK




SSLQPEDFATYYC



807B-M0079-H05
GVPSRFSGSGSGTLFTLTI
QQYQNYPYT
FGQGTKLDIE




NNLQPEDFATYYC



807B-M0080-A02
GVPSRFSGSGSGTDFTLTI
QQSYTTPLT
FGGGTKVEIK




SSLQPEDFATYFC



807B-M0080-C04
GVPARFSGSKSGTSASLAI
AAWDDSLNGPNVV
FGGGTKLTVL




SGLQSEDEADYYC



807B-M0080-F10
GAPSRFSGSGSGTDFTLTI
QQANSFPLT
FGGGTKVEIK




SSLQPEDFATYYC



807B-M0081-C03
GAPSRFSGSGSGTEFTLTI
QQYHSYPWT
FGQGTKVDVK




SSLQPDDFGTYYC



807B-M0081-C05
GVPDRFSGSKSGTSASLAI
QSYDSSLSGPI
FGGGTTLTVL




SGLQAEDEAHYYC



807B-M0081-D08
GVPSRFSGSGSGTDFTLTI
QQANSFPPT
FGQGTKVEIQ




SSLQPEDFATYYC



807B-M0081-E08
GVPPRFSGSGSGTDFSLII
QQSYAAPLT
FGGGTKVEIK




SSLQPEDFATYYC



807B-M0081-F12
GVPSRFSGSGSGTEFSLSI
QQANSFPLT
FGGGTKVEIK




SSLQPEDFATYYC



807B-M0081-G04
GVPARFSGTGSGTDFTLTI
QQRSNWPYT
FGQGTKLEIK




SSLEPEDFAVYYC



807B-M0081-G11
GVPSRFSGSGTGTDFTLTI
QQSYSSPGIT
FGPGTKVEIK




RSLQPEDFASYFC



807B-M0081-H03
GVPDRFSGSKSGNTASLT
SSYAGRNFVV
FGGGTKLTVL




VSGLQAEDEADYYC



807B-M0081-H07
GVPSRFSGSGSGTDFTLII
QQSYTTPFT
FGPGTTVDIK




SDLQPEDFATYYC



807B-M0082-B07
GVSNRFSGSKSGNTASLTI
SSYTSRSTYV
FGTGTKVTVL




SGLQAEDEADYYC



807B-M0082-E01
GVPSRFSGSGSGTDFTLT
LQSNTFPFT
FGPGTKVDIT




VSSLQPEDFATYYC



807B-M0082-E08
GVPSRFSGSGSGTDFTLTI
QQTYSSPWT
FGQGTKVEIR




SNLQPEDFATYYC



807B-M0082-H06
GVPSRFSGSGSGTEFTLTI
LQHNNYPYS
FGQGTKLEIK




SSLQPEDFATYYC



807B-M0083-B10
GVPSRFSGSGSGTDFTLTI
QQSYSTPRT
FGQGTKVEIK




SSLQPEDFATYYC



807B-M0083-E10
GIPARFSGSGSGTDFTLTI
QQSYEYPLT
FGQGTKLEIK




SSLQPEDFATYYC



807B-M0083-E11
GIPERFSGSNSGNTATLTI
QLWDSSSDHPI
FGTGTKVTVL




SRVEAGDEADYYC



807B-M0084-C03
GIPERFFGSNSGRTATLTIS
QVWDSSSDDYV
FAAGTKLSVL




GVEVGDEADYYC



807B-M0084-C11
GVPSRFSGSGSGTDFTLTI
QHSYGTPT
FGQGTKVEIK




SSLQPEDFATYYC



807B-M0084-E07
GVPSRFSGSGSGTDFTLTI
QQSYITPRT
FGQGTKVEIK




SNLQPEDFATYYC



807B-M0084-F03
GIPARFSGSGSGTDFTLTI
QQYYYTPPT
FGRGTKVEIN




SSLEPEDFAVYYC



807B-M0084-F08
DIPARFSGGGSGTEFTLTI
QHEET
FGQGTKVEIK




SSLQPDDFATYYC



807B-M0084-H05
GVSDRFSGSKSGTSASLAI
AAWDDSLSGVV
FGGGTKLTVL




SGLRSEDEADYYC



807B-M0085-B12
GVPSRFSGSGSGTDFTLTI
QQANSFPLT
FGGGTKVEIK




SSLQPEDFATYYC



807B-M0085-C01
GIPARFSGSGSGTDFTFTI
QQYINLPIT
FGQGTRLEIK




SSLQPEDIATYFC



807B-M0085-E10
GVPSRFSGSGSGTHFTFTI
QQFDNFPLT
FGPGTRLDIK




SSLQPEDFATYYC



807B-M0085-G03
GVPSRFSGSGSGTDFTLTI
QQSYSTPLYT
FGQGTKLEIK




SSLQPEDFATYYC



807B-M0085-G07
GIPDRFSGSGSGTDFTLTI
QSGVT
FGGGTKVEIK




SRLEPEDFAVYYC



807B-M0085-G08
GIPDRFSGSGSGTDFTLTI
QQYGNSPGGT
FGQGTKVEIK




SRLEPEDFAVYYC



807B-M0086-C06
GVPDRFSGSKSGTSASLAI
AAWDDSLSGGV
FGGGTKLTVL




SGLRSEDEADYYC



807B-M0086-D03
GVPSRFSGSGSGTDFTLTI
QQSYSIPPT
FGQGTRVEIK




SSLQPEDFATYYC



807B-M0086-E08
GIPDRFSGSGSRTDFTLTI
QQYVTTPPT
FGQGTKVEIK




SRLEPEDFAVYYC



807B-M0086-G03
GVPSRFSAGGSGTEFTLTI
QQVSSYPLT
FGGGPRVEIK




NSLQAEDFATYYC

















TABLE 15





Amino acid sequences of the VH chains of the antibodies


identified using the screening strategy of Example 23



















Isolate Name
FR1
CDR1
FR2
CDR2





807B-M0011-C07
EVQLLESGGGLVQPG
WYSMD
WVRQAPGKGLEWVS
GIGPSGGRTRY



GSLRLSCAASGFTFS


ADSVKG


807B-M0012-C09
EVQLLESGGGLVQPG
WYTMD
WVRQAPGKGLEWVS
GISPSGGATNY



GSLRLSCAASGFTFS


ADSVKG


807B-M0012-D09
EVQLLESGGGLVQPG
LYQMA
WVRQAPGKGLEWVS
SISSSGGLTDY



GSLRLSCAASGFTFS


ADSVKG


807B-M0012-F10
EVQLLESGGGLVQPG
WYNMS
WVRQAPGKGLEWVS
YIYPSGGITIYA



GSLRLSCAASGFTFS


DSVKG


807B-M0012-F12
EVQLLESGGGLVQPG
WYDMD
WVRQAPGKGLEWVS
SIYPSGGLTGY



GSLRLSCAASGFTFS


ADSVKG


807B-M0012-G05
EVQLLESGGGLVQPG
WYRMT
WVRQAPGKGLEWVS
SISPSGGVTLYA



GSLRLSCAASGFTFS


DSVKG


807B-M0013-A12
EVQLLESGGGLVQPG
HYGMS
WVRQAPGKGLEWVS
SIRSSGGRTWY



GSLRLSCAASGFTFS


ADSVKG


807B-M0013-B04
EVQLLESGGGLVQPG
HYDMV
WVRQAPGKGLEWVS
VIVPSGGATAY



GSLRLSCAASGFTFS


ADSVKG


807B-M0013-C03
EVQLLESGGGLVQPG
WYNMA
WVRQAPGKGLEWVS
SISPSGGHTKY



GSLRLSCAASGFTFS


ADSVKG


807B-M0013-F06
EVQLLESGGGLVQPG
KYVMT
WVRQAPGKGLEWVS
VISSSGGPTDY



GSLRLSCAASGFTFS


ADSVKG


807B-M0013-G05
EVQLLESGGGLVQPG
WYSMI
WVRQAPGKGLEWVS
YIGPSGGPTRY



GSLRLSCAASGFTFS


ADSVKG


807B-M0014-D07
EVQLLESGGGLVQPG
WYTMT
WVRQAPGKGLEWVS
SISSSGGVTKY



GSLRLSCAASGFTFS


ADSVKG


807B-M0014-D09
EVQLLESGGGLVQPG
YYIMA
WVRQAPGKGLEWVS
SISPSGGGTVY



GSLRLSCAASGFTFS


ADSVKG


807B-M0014-E08
EVQLLESGGGLVQPG
WYAMD
WVRQAPGKGLEWVS
SIYPSGGWTEY



GSLRLSCAASGFTFS


ADSVKG


807B-M0014-F07
EVQLLESGGGLVQPG
WYDMF
WVRQAPGKGLEWVS
SISPSGGFTQY



GSLRLSCAASGFTFS


ADSVKG


807B-M0016-C06
EVQLLESGGGLVQPG
LYGMS
WVRQAPGKGLEWVS
SIGPSGGHTFY



GSLRLSCAASGFTFS


ADSVKG


807B-M0016-D01
EVQLLESGGGLVQPG
WYNMG
WVRQAPGKGLEWVS
GISPSGGTTTY



GSLRLSCAASGFTFS


ADSVKG


807B-M0016-D08
EVQLLESGGGLVQPG
WYGMV
WVRQAPGKGLEWVS
SIYPSGGTTQY



GSLRLSCAASGFTFS


ADSVKG


807B-M0016-E01
EVQLLESGGGLVQPG
WYDMQ
WVRQAPGKGLEWVS
SISSSGGLTTYA



GSLRLSCAASGFTFS


DSVKG


807B-M0016-F04
EVQLLESGGGLVQPG
NYNMH
WVRQAPGKGLEWVS
VISPSGGGTWY



GSLRLSCAASGFTFS


ADSVKG


807B-M0016-F05
EVQLLESGGGLVQPG
WYRMT
WVRQAPGKGLEWVS
SISPSGGVTLYA



GSLRLSCAASGFTFS


DSVKG


807B-M0016-F08
EVQLLESGGGLVQPG
MYHMG
WVRQAPGKGLEWVS
GISPSGGTTTY



GSLRLSCAASGFTFS


ADSVKG


807B-M0017-B05
EVQLLESGGGLVQPG
WYTMF
WVRQAPGKGLEWVS
GIWPSGGKTDY



GSLRLSCAASGFTFS


ADSVKG


807B-M0017-B06
EVQLLESGGGLVQPG
WYEMG
WVRQAPGKGLEWVS
SISPSGGYTSY



GSLRLSCAASGFTFS


ADSVKG


807B-M0017-E05
EVQLLESGGGLVQPG
WYQMA
WVRQAPGKGLEWVS
GISSSGGTTTY



GSLRLSCAASGFTFS


ADSVKG


807B-M0018-C12
EVQLLESGGGLVQPG
FYRMG
WVRQAPGKGLEWVS
SISSSGGLTDY



GSLRLSCAASGFTFS


ADSVKG


807B-M0018-E09
EVQLLESGGGLVQPG
WYGMA
WVRQAPGKGLEWVS
YISPSGGGTSY



GSLRLSCAASGFTFS


ADSVKG


807B-M0018-G02
EVQLLESGGGLVQPG
KYVMN
WVRQAPGKGLEWVS
SISSSGGQTSY



GSLRLSCAASGFTFS


ADSVKG


807B-M0019-A04
EVQLLESGGGLVQPG
YYDMW
WVRQAPGKGLEWVS
RIVSSGGWTMY



GSLRLSCAASGFTFS


ADSVKG


807B-M0019-A10
EVQLLESGGGLVQPG
YYDMS
WVRQAPGKGLEWVS
SIWSSGGNTMY



GSLRLSCAASGFTFS


ADSVKG


807B-M0019-C01
EVQLLESGGGLVQPG
WYGMG
WVRQAPGKGLEWVS
YISSSGGHTKY



GSLRLSCAASGFTFS


ADSVKG


807B-M0019-F06
EVQLLESGGGLVQPG
WYPMD
WVRQAPGKGLEWVS
SISPSGGFTQY



GSLRLSCAASGFTFS


ADSVKG


807B-M0019-G07
EVQLLESGGGLVQPG
FYPMV
WVRQAPGKGLEWVS
WISSSGGTTSY



GSLRLSCAASGFTFS


ADSVKG


807B-M0020-D01
EVQLLESGGGLVQPG
WYVMT
WVRQAPGKGLEWVS
GISSSGGMTEY



GSLRLSCAASGFTFS


ADSVKG


807B-M0020-F06
EVQLLESGGGLVQPG
VYNMY
WVRQAPGKGLEWVS
SISPSGGFTTYA



GSLRLSCAASGFTFS


DSVKG


807B-M0020-F12
EVQLLESGGGLVQPG
IYEMA
WVRQAPGKGLEWVS
SISPSGGWTKY



GSLRLSCAASGFTFS


ADSVKG


807B-M0020-G01
EVQLLESGGGLVQPG
FYYMS
WVRQAPGKGLEWVS
GISPSGGTTQY



GSLRLSCAASGFTFS


ADSVKG


807B-M0079-B09
EVQLLESGGGLVQPG
FYAMQ
WVRQAPGKGLEWVS
YISSSGGHTHY



GSLRLSCAASGFTFS


ADSVKG


807B-M0079-D10
EVQLLESGGGLVQPG
NYRME
WVRQAPGKGLEWVS
SIWSSGGLTKQ



GSLRLSCAASGFTFS


ADSVKG


807B-M0079-H01
EVQLLESGGGLVQPG
WYDMD
WVRQAPGKGLEWVS
RIWPSGGSTHY



GSLRLSCAASGFTFS


ADSVKG


807B-M0079-H05
EVQLLESGGGLVQPG
QYMMG
WVRQAPGKGLEWVS
SISSGGWTAYA



GSLRLSCAASGFTFS


DSVKG


807B-M0080-A02
EVQLLESGGGLVQPG
WYNMG
WVRQAPGKGLEWVS
SIGPSGGHTMY



GSLRLSCAASGFTFS


ADSVKG


807B-M0080-C04
EVQLLESGGGLVQPG
WYSME
WVRQAPGKGLEWVS
SIVSSGGHTIYA



GSLRLSCAASGFTFS


DSVKG


807B-M0080-F10
EVQLLESGGGLVQPG
DYVMN
WVRQAPGKGLEWVS
SIYPSGGLTRY



GSLRLSCAASGFTFS


ADSVKG


807B-M0081-C03
EVQLLESGGGLVQPG
WYWMG
WVRQAPGKGLEWVS
GISSSGGRTVY



GSLRLSCAASGFTFS


ADSVKG


807B-M0081-C05
EVQLLESGGGLVQPG
WYNMD
WVRQAPGKGLEWVS
SIGPSGGPTKY



GSLRLSCAASGFTFS


ADSVKG


807B-M0081-D08
EVQLLESGGGLVQPG
EYTML
WVRQAPGKGLEWVS
GIWPSGGPTFY



GSLRLSCAASGFTFS


ADSVKG


807B-M0081-E08
EVQLLESGGGLVQPG
MYNMY
WVRQAPGKGLEWVS
RIGSSGGMTDY



GSLRLSCAASGFTFS


ADSVKG


807B-M0081-F12
EVQLLESGGGLVQPG
WYLMH
WVRQAPGKGLEWVS
SIVPSGGTTVY



GSLRLSCAASGFTFS


ADSVKG


807B-M0081-G04
EVQLLESGGGLVQPG
WYSMV
WVRQAPGKGLEWVS
VISSSGGFTGY



GSLRLSCAASGFTFS


ADSVKG


807B-M0081-G11
EVQLLESGGGLVQPG
WYDMV
WVRQAPGKGLEWVS
GIWPSGGFTNY



GSLRLSCAASGFTFS


ADSVKG


807B-M0081-H03
EVQLLESGGGLVQPG
WYSMV
WVRQAPGKGLEWVS
SIGPSGGMTRY



GSLRLSCAASGFTFS


ADSVKG


807B-M0081-H07
EVQLLESGGGLVQPG
FYTMV
WVRQAPGKGLEWVS
VISPSGGLTHY



GSLRLSCAASGFTFS


ADSVKG


807B-M0082-B07
EVQLLESGGGLVQPG
YYSMT
WVRQAPGKGLEWVS
SISPSGGGTGY



GSLRLSCAASGFTFS


ADSVKG


807B-M0082-E01
EVQLLESGGGLVQPG
WYGMS
WVRQAPGKGLEWVS
WISPSGGMTKY



GSLRLSCAASGFTFS


ADSVKG


807B-M0082-E08
EVQLLESGGGLVQPG
WYDMD
WVRQAPGKGLEWVS
SISSSGGFTTYA



GSLRLSCAASGFTFS


DSVKG


807B-M0082-H06
EVQLLESGGGLVQPG
WYGMG
WVRQAPGKGLEWVS
YISSSGGLTIYA



GSLRLSCAASGFTFS


DSVKG


807B-M0083-B10
EVQLLESGGGLVQPG
YYAMG
WVRQAPGKGLEWVS
WISPSGGATHY



GSLRLSCAASGFTFS


ADSVKG


807B-M0083-E10
EVQLLESGGGLVQPG
YYSMV
WVRQAPGKGLEWVS
WISSSGGSTNY



GSLRLSCAASGFTFS


ADSVKG


807B-M0083-E11
EVQLLESGGGLVQPG
VYSMA
WVRQAPGKGLEWVS
GIWPSGGPTAY



GSLRLSCAASGFTFS


ADSVKG


807B-M0084-C03
EVQLLESGGGLVQPG
WYDMM
WVRQAPGKGLEWVS
YIYSSGGSTRY



GSLRLSCAASGFTFS


ADSVKG


807B-M0084-C11
EVQLLESGGGLVQPG
FYRMA
WVRQAPGKGLEWVS
VISPSGGHTYY



GSLRLSCAASGFTFS


ADSVKG


807B-M0084-E07
EVQLLESGGGLVQPG
WYRMA
WVRQAPGKGLEWVS
SISSSGGDTQY



GSLRLSCAASGFTFS


ADSVKG


807B-M0084-F03
EVQLLESGGGLVQPG
WYVMD
WVRQAPGKGLEWVS
SISPSGGGTLY



GSLRLSCAASGFTFS


ADSVKG


807B-M0084-F08
EVQLLESGGGLVQPG
WYGMA
WVRQAPGKGLEWVS
YISPSGGGTSY



GSLRLSCAASGFTFS


ADSVKG


807B-M0084-H05
EVQLLESGGGLVQPG
WYTME
WVRQAPGKGLEWVS
GIYSSGGTTTY



GSLRLSCAASGFTFS


ADSVKG


807B-M0085-B12
EVQLLESGGGLVQPG
WYGMV
WVRQAPGKGLEWVS
SISPSGGQTFY



GSLRLSCAASGFTFS


ADSVKG


807B-M0085-C01
EVQLLESGGGLVQPG
WYTMD
WVRQAPGKGLEWVS
GISPSGGYTTY



GSLRLSCAASGFTFS


ADSVKG


807B-M0085-E10
EVQLLESGGGLVQPG
WYQMS
WVRQAPGKGLEWVS
GISSSGGSTQY



GSLRLSCAASGFTFS


ADSVKG


807B-M0085-G03
EVQLLESGGGLVQPG
WYVMM
WVRQAPGKGLEWVS
SIVPSGGGTGY



GSLRLSCAASGFTFS


ADSVKG


807B-M0085-G07
EVQLLESGGGLVQPG
WYAMD
WVRQAPGKGLEWVS
SIVPSGGRTLY



GSLRLSCAASGFTFS


ADSVKG


807B-M0085-G08
EVQLLESGGGLVQPG
FYGMG
WVRQAPGKGLEWVS
RIRPSGGMTSY



GSLRLSCAASGFTFS


ADSVKG


807B-M0086-C06
EVQLLESGGGLVQPG
WYSMV
WVRQAPGKGLEWVS
WISSSGGFTKY



GSLRLSCAASGFTFS


ADSVKG


807B-M0086-D03
EVQLLESGGGLVQPG
WYTMG
WVRQAPGKGLEWVS
SIVSSGGYTPY



GSLRLSCAASGFTFS


ADSVKG


807B-M0086-E08
EVQLLESGGGLVQPG
WYDMH
WVRQAPGKGLEWVS
WIVPSGGITEYA



GSLRLSCAASGFTFS


DSVKG


807B-M0086-G03
EVQLLESGGGLVQPG
EYKMN
WVRQAPGKGLEWVS
YIYPSGGFTHY



GSLRLSCAASGFTFS


ADSVKG













Isolate Name
FR3
CDR3
FR4





807B-M0011-C07
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0012-C09
RFTISRDNSKNTLYLQMNSL
GLRYFDFYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0012-D09
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0012-F10
RFTISRDNSKNTLYLQMNSL
ERGTIFNDAFDI
WGQGTMVTVSS



RAEDTAVYYCAR


807B-M0012-F12
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0012-G05
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0013-A12
RFTISRDNSKNTLYLQMNSL
GSLSSGWDY
WGQGTLVTVSS



RAEDTAVYYCAK


807B-M0013-B04
RFTISRDNSKNTLYLQMNSL
EDFWSGLEDV
WGKGTTVTVSS



RAEDTAVYYCAR


807B-M0013-C03
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0013-F06
RFTISRDNSKNTLYLQMNSL
WGVRGVIPFDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0013-G05
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0014-D07
RFTISRDNSKNTLYLQMNSL
GRWLAPFDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0014-D09
RFTISRDNSKNTLYLQMNSL
ATCTGGSCYRFDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0014-E08
RFTISRDNSKNTLYLQMNSL
GLGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0014-F07
RFTISRDNSKNTLYLQMNSL
QEVWQWPAQFDS
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0016-C06
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0016-D01
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0016-D08
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0016-E01
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0016-F04
RFTISRDNSKNTLYLQMNSL
DFFTSYFDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0016-F05
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0016-F08
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0017-B05
RFTISRDNSKNTLYLQMNSL
EGVIAVAGPYRD
WGQGTLVTVSS



RAEDTAVYYCAK


807B-M0017-B06
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0017-E05
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0018-C12
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0018-E09
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0018-G02
RFTISRDNSKNTLYLQMNSL
GERAAAGTQHYYYY
WGQGTTVTVSS



RAEDTAVYYCAR
YGMDV


807B-M0019-A04
RFTISRDNSKNTLYLQMNSL
GRSLYYDFWSGYYP
WGKGTTVTVSS



RAEDTAVYYCAR
NTYYYYYMDV


807B-M0019-A10
RFTISRDNSKNTLYLQMNSL
GGGFGVYHHYYDM
WGQGTTVTVSS



RAEDTAVYYCAR
DV


807B-M0019-C01
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0019-F06
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0019-G07
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0020-D01
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0020-F06
RFTISRDNSKNTLYLQMNSL
DTSGWYEEEDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0020-F12
RFTISRDNSKNTLYLQMNSL
DLWFGEWDH
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0020-G01
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0079-B09
RFTISRDNSKNTLYLQMNSL
GAGALGY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0079-D10
RFTISRDNSKNTLYLQMNSL
GLYR
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0079-H01
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0079-H05
RFTISRDNSKNTLYLQMNSL
DGGTWDFDY
WGQGTLVTVSS



RAEDTAVYYCAK


807B-M0080-A02
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0080-C04
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0080-F10
RFTISRDNSKNTLYLQMNSL
GAGALGY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0081-C03
RFTISRDNSKNTLYLQMNSL
GHWGWFDP
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0081-C05
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0081-D08
RFTISRDNSKNTLYLQMNSL
ELDTAMAPPSDAFDI
WGQGTMVTVSS



RAEDTAVYYCAR


807B-M0081-E08
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0081-F12
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY



RAEDTAVYYCAR


807B-M0081-G04
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0081-G11
RFTISRDNSKNTLYLQMNSL
HPVSSGFDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0081-H03
RFTISRDNSKNTLYLQMNSL
DQGITMVQGAMGY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0081-H07
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0082-B07
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0082-E01
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0082-E08
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0082-H06
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0083-B10
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0083-E10
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0083-E11
RFTISRDNSKNTLYLQMNSL
EDFWSGLEDV
WGKGTTVTVSS



RAEDTAVYYCAR


807B-M0084-C03
RFTISRDNSKKTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0084-C11
RFTISRDNSKNTLYLQMNSL
DFFTSYFDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0084-E07
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0084-F03
RFTISRDNSKNTLYLQMNSL
DLHYGSVLDF
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0084-F08
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0084-H05
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0085-B12
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0085-C01
RFTISRDNSKNTLYLQMNSL
GTVLLWFGESGGHF
WGQGTPVTVSS



RAEDTAVYYCAR
DY


807B-M0085-E10
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0085-G03
RFTISRDNSKNTLYLQMNSL
ENYGPDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0085-G07
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0085-G08
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0086-C06
RFTISRDNSKNTLYLQMNSL
DLWFGEWDY
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0086-D03
RFTISRDNSKNTLYLQMNSL
EPIWGYYYYGMDV
WGQGTTVTVSS



RAEDTAVYYCAR


807B-M0086-E08
RFTISRDNSKNTLYLQMNSL
QEVWQWPAQFDS
WGQGTLVTVSS



RAEDTAVYYCAR


807B-M0086-G03
RFTISRDNSKNTLYLQMNSL
SVVGWGLDY
WGQGTLVTVSS



RAEDTAVYYCAR























TABLE 16









Enrichment

Peptide mapping
koff

Fab
























Isolate Name
VH
CDR3
bCTD
VLDL
1
2
3
4
5
6
7
8
9
10
(e−3)
RU
IHC



























807B-M0011-C07
3
20
+











11.6
325



807B-M0012-C09
12
12
+











11.7
156


807B-M0012-D09
1
299
+











14.8
159


807B-M0012-F10
1
1
+











5.5
50.5


807B-M0012-F12
2
20
+











3.0
246


807B-M0012-G05
15
299
+











7.9
157


807B-M0013-A12
3
3
+











6.8
63.9
+


807B-M0013-B04
1
4
+











12.5
101


807B-M0013-C03
1
299
+



+







38.4
177


807B-M0013-F06
1
1
+



+




+


9.3
162


807B-M0013-G05
1
20
+
+/−


+







17.5
136


807B-M0014-D07
10
10
+








+


8.1
206


807B-M0014-D09
13
13
+








+


5.2
80.4


807B-M0014-E08
2
2
+



+







18.6
151


807B-M0014-F07
1
2
+











8.6
205


807B-M0016-C06
5
299
+











7.2
74.2


807B-M0016-D01
1
299
+











8.5
74


807B-M0016-D08
1
299
+



+







12.4
80.3


807B-M0016-E01
1
20
+











8.0
465


807B-M0016-F04
1
2
+











13.5
58.9


807B-M0016-F05
15
299
+











9.0
69


807B-M0016-F08
1
299
+











18.5
113


807B-M0017-B05
1
1
+
+/−










26.1
248


807B-M0017-B06
1
299
+











8.8
120


807B-M0017-E05
1
299
+











4.7
71.1


807B-M0018-C12
2
299
+











11.0
107


807B-M0018-E09
3
299
+











10.5
66.8


807B-M0018-G02
1
1
+








+


21.1
98.9


807B-M0019-A04
1
1
+











5.4
31.1


807B-M0019-A10
1
1
+











7.1
51


807B-M0019-C01
1
299
+











10.6
80.1


807B-M0019-F06
1
299
+











7.7
99.4


807B-M0019-G07
1
299
+











5.2
31.2


807B-M0020-D01
1
299
+











3.9
51.2


807B-M0020-F06
1
1
+











4.9
53.2


807B-M0020-F12
1
1
+











9.9
67.4


807B-M0020-G01
1
299
+











12.7
68


807B-M0079-B09
1
2
+
+/+






+



8.3
438


807B-M0079-D10


+




+



+
+

4.3
100
+


807B-M0079-H01
1
20
+



+
+
+





2.5
256


807B-M0079-H05
1
1
+











2.0
99.3


807B-M0080-A02
1
299
+











2.5
86.8


807B-M0080-C04
1
20
+











2.5
162


807B-M0080-D06
1
1
+





+





2.4
72.7


807B-M0080-F10
1
2
+






+




2.7
49.2


807B-M0081-C03
1
1
+








+


2.2
84.6


807B-M0081-C05
1
20
+











2.6
158


807B-M0081-D08
1
1
+






+




4.0
55.1


807B-M0081-E08
1
20
+











5.7
65.1


807B-M0081-F12
249
299
+











6.1
128
+


807B-M0081-G04
1
299
+











2.6
64.1


807B-M0081-G11
1
1
+






+




3.6
83.6


807B-M0081-H03
1
1
+











9.7
346
+/−


807B-M0081-H07
1
299
+











2.3
56.4


807B-M0082-B07
1
299
+
+/−










2.8
46.4


807B-M0082-C11
1
1
+











4.0
244


807B-M0082-E01
2
299
+





+
+




4.7
68.8


807B-M0082-E08
1
20
+











3.0
154


807B-M0082-F04
1
20
+











3.1
136


807B-M0082-F11
1
299
+











6.1
32.5


807B-M0082-H06
2
299
+




+






5.1
103


807B-M0083-B10
1
299
+





+
+




2.9
45


807B-M0083-E10
1
299
+





+





3.7
47.9


807B-M0083-E11
3
4
+











5.1
34.6
+


807B-M0084-C03
1
20
+











2.0
53.9


807B-M0084-C11
1
2
+











3.7
52.5


807B-M0084-E07
1
299
+

+



+

+



4.4
46.7


807B-M0084-F03
1
1
+











3.6
49.6


807B-M0084-F08
3
299
+











5.8
51.5


807B-M0084-H05
2
20
+
+










2.4
118


807B-M0085-B12
1
20
+





+





7.9
95.3


807B-M0085-C01
1
1
+











5.5
69.7


807B-M0085-E01
1
20
+











2.8
49.5


807B-M0085-E10
1
299
+

+
+


+
+

+
+
+
5.8
47.5


807B-M0085-G03
1
1
+











2.2
52.8


807B-M0085-G07
1
20
+











4.2
83.7


807B-M0085-G08
1
299
+









+

3.8
42.2


807B-M0086-C06
1
299
+











4.8
59.5


807B-M0086-D03
1
20
+











3.7
159


807B-M0086-E08
1
2
+











2.6
73


807B-M0086-G03
2
2
+











3.7
43.8
















TABLE 17







Fab Binding Data to CTD and peptides












CTD

Affinity Biacore (nM)
VLDL



















Isolate name
Strategy
IHC
ELISA
peptide
group
CTD
p1
p4
p6
p8
p9
ELISA






















807B-M0001-B07
E22 P1
+/−
+
p1, p6
4
46
245

396


−


807B-M0004-A03
E22 P4
++
+
p4, p9
5
low

98


low
−








RU*




RU*


807B-M0004-A05
E22 P4
+/−
−
p4
1
no

208



−








RU**


807B-M0004-C04
E22 P4
+/−
−
p4
1
no

509



−








RU**


807B-M0004-C05
E22 P4
+/−
−
p4
1
no

225



−








RU**


807B-M0004-F06
E22 P4
+/−
−
p4
1
no

361



−








RU**


807B-M0004-F10
E22 P4
+/−
−
p4, p9
2
no

104


low
−








RU**




RU*


807B-M0004-H03
E22 P4
++
−
p4, p9
2
no

200


low
−








RU**




RU*


807B-M0009-C03
E22 P9,
+/−
+
p4, p9
5
no

728


163
−



E24 P3




RU**


807B-M0009-F06
E22 P9,
+/−
−
p9
3
no




172
−



E24 P3




RU**


807B-M0013-A12
E24
++
+
no
7
no





−








RU**


807B-M0079-D10
E22&E23
++
+
p4, p8
6
59.17

low

25.79

−



P9, E24






RU*


807B-M0081-F12
E24
+
+
no
8
low





−








RU*


807B-M0081-H03
E24
+/−
+
no
9
10





−


807B-M0083-E11
E24
++
+
no
10
low





−








RU*


807A-M0028-B02
E5
+
+
no







−


807A-M0026-F05
E5
+
+
p3, p8







−


807A-M0027-E11
E5
+
+
no







−





*low RU: RUs are low, affinity was not measured


**no RU: no binding observed













TABLE 18





IgG Binding Data to CTD and peptides


















CTD
Affinity Biacore (nM)














Isolate name
Strategy
IHC
ELISA
peptide
hCTD
mCTD
pCTD





807B-M0001-B07
E22 P1
+
+
p1, p6
5
<h, pCTD
6


807B-M0004-A03
E22 P4
+++
+
(p1, p6)
3

3






p4, p8, p9


807B-M0004-A05
E22 P4
−
+
p4, p9
167
no RU**
no RU**


807B-M0004-C04
E22 P4
−
+/−
p4, p9
no RU**
no RU**
no RU**


807B-M0004-C05
E22 P4
−
−
p4, p9
no RU**
no RU**
no RU**


807B-M0004-F06
E22 P4
−


no RU**
no RU**
no RU**


807B-M0004-F10
E22 P4
(+)
+
p4, p8, p9
25
no RU**
23


807B-M0004-H03
E22 P4
+(+)
+
p4, p9,
45
no RU**
43






(p8)


807B-M0009-C03
E22 P9,
(+)
+
p4, p9
23
no RU**
4



E24 P3


807B-M0009-F06
E22 P9,
++
−
p4, p9
34
234
28



E24 P3


807B-M0013-A12
E24
+++
+
(p3, p9),
27
23
26






p4, p8


807B-M0079-D10
E22&E23
(+)
+
p4, p8
1
2
1



P9, E24


807B-M0081-F12
E24

+
(p9)
15
16
19


807B-M0081-H03
E24
(+)
+
(p7)
9
18
7


807B-M0083-E11
E24
−
+
no
12
64
25


807A-M0028-B02
E5
+
+
P4
9
7
5


807A-M0026-F05
E5
+


807A-M0027-E11
E5
+















VLDL




Affinity Biacore (nM)
ELISA
ELISA















Isolate name
p1
p4
p8
p9
(O.D)†
hCTD
mCTD
pCTD





807B-M0001-B07
8.04



2.14
+++
++
+++


807B-M0004-A03

0.17

low RU*
0.273
+
+
++


807B-M0004-A05

17.42


0.214
+/−
+/−
+/−


807B-M0004-C04

8.68


0.242
+/−
+/−
+/−


807B-M0004-C05

0.97


0.233
+/−
+/−
+/−


807B-M0004-F06

6.72


0.248
+/−
+/−
+/−


807B-M0004-F10

1.00

13.74
1.11
++
+/−
+/−


807B-M0004-H03

11.55

low RU*
0.309
++
+(+)
++


807B-M0009-C03

1.00

0.27
2.528
+++
+++
+++


807B-M0009-F06

no RU**

10.6
1.202
+/−
+/−
+/−


807B-M0013-A12




0.462
++
+/−
+/−


807B-M0079-D10

43.64
1.05

0.2
+++
+++
+++


807B-M0081-F12

no RU**


0.285
++
+
+


807B-M0081-H03

no RU**


2.88
+++
+++
+++


807B-M0083-E11

no RU**


0.221
+
−
−


807A-M0028-B02




2.679


807A-M0026-F05







−


807A-M0027-E11







−





*low RU: RUs are low, affinity was not measured


**no RU: no binding observed


†VLDL ELISA: VLDL is coated, 5 μg/ml of appropriate hIgG is added O.D. of 2x background is 0.420













TABLE 19





Amino acid sequences of the VL chains of the Germline-corrected antibodies



















Initial Name
LV-FR1
LV-CDR1
LV-FR2
LV-CDR2





807A-M0028-B02.1
DIQMTQSPSSLSA
RTSQDIRNHLG
WFQQKPGKAP
EASILQS



SVGDRVTITC

QRLIR


807A-M0028-B02.2
DIQMTQSPSSLSA
RTSQDIRNHLG
WYQQKPGKAP
EASILQS



SVGDRVTITC

KRLIY


807B-M0004-H03.1
DIQMTQSPSSLSA
QASQNIDNYLN
WYQQKPGKAP
AASSLQS



SVGDRVTITC

KLLIY


807B-M0009-F06.1
DIVMTQSPLSLPV
KSSQSLLHSNGYNYLD
WYLQKPGQSP
LGSNRAS



TPGEPASISC

QLLIY


807B-M0004-A03.1
QSVLTQPPSASG
SGSSSNIGSNTVN
WYQQLPGTAP
NNNQRPS



TPGQRVTISC

KLLIY


807B-M0079-D10.1
SYELTQPPSVSVS
AGDELGNKYAS
WYQQKPGQSP
QDRKRPS



PGQTASITC

VLVIY















Initial Name
LV-FR3
LV-CDR3
LV-FR4







807A-M0028-B02.1
GVPSTFYGSGYGREFTLTIS
LQYDSFPYT
FGQGTKLEIK




SLQPEDFATYYC



807A-M0028-B02.2
GVPSRFSGSGSGTEFTLTIS
LQYDSFPYT
FGQGTKLEIK




SLQPEDFATYYC



807B-M0004-H03.1
GVPSRFSGSGSGTDFTLTIS
QQSYSTPRT
FGQGTKVEIK




SLQPEDFATYYC



807B-M0009-F06.1
GVPDRFSGSGSGTDFTLKIS
MQALQTIT
FGQGTRLEIK




RVEAEDVGVYYC



807B-M0004-A03.1
GVPDRFSGSKSGTSASLAIS
AAWHDGLNGPV
FGGGTKLTVL




GLQSEDEADYYC



807B-M0079-D10.1
GIPERFSGSNSGNTATLTIS
QSWDSSSVI
FGGGTKLTVL




GTQAMDEADYYC

















TABLE 20







Amino acid sequences of the CL chains of the Germline-corrected antibodies









Name
Germ line
Sequence of constant region of light chain





807A-M0028-B02.1
Kappa
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL




SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC


807A-M0028-B02.2
Kappa
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL




SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC


807B-M0004-A03.1
Lambda
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL




TPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS


807B-M0079-D10.1
Lambda
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL




TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
















TABLE 21







Description of SEQ ID NOS: 539-548











Antibody
VL chain sequence
CL chain sequence







807A-M0028-B02.1
SEQ ID NO: 518
SEQ ID NO: 519



807A-M0028-B02.2
SEQ ID NO: 520
SEQ ID NO: 521



807B-M0004-H03.1
SEQ ID NO: 524
—



807B-M0009-F06.1
SEQ ID NO: 525
—



807B-M0004-A03.1
SEQ ID NO: 522
SEQ ID NO: 523



807B-M0079-D10.1
SEQ ID NO: 526
SEQ ID NO: 527

















TABLE 22







IgG Binding Data to CTD and peptides











Isolate
CTD
Affinity Biacore (nM)
VLDL
ELISA6






















name
Strategy
IHC2
ELISA
peptide
hCTD
mCTD
pCTD
p1
p4
p8
p9
ELISA5
hCTD
mCTD
pCTD

























807B-
E22 P1
+
+
p1, p6
5
<h, pCTD
6
8.04



++
+++
++
+++


M0001-


B07


807B-
E22 P4
+++
+
(p1, p6)
3

3

0.17

low
++
+++
++
+


M0004-



p4, p8,






RU4


A03



P9


807B-
E22 P4
−
+
p4, p9
167
no RU3
no RU3

17.42


−
+/−
+/−
+/−


M0004-


A05


807B-
E22 P4
−
+/−
p4, p9
no RU3
no RU3
no RU3

8.68


−
+/−
+/−
+/−


M0004-


C04


807B-
E22 P4
−
−
p4, p9
no RU3
no RU3
no RU3

0.97


−
+/−
+/−
+/−


M0004-


C05


807B-
E22 P4
−


no RU3
no RU3
no RU3

6.72


−
+/−
+/−
+/−


M0004-


F06


807B-
E22 P4
(+)
+
p4, p8,
25
no RU3
23

1.00

13.74
+
++
+/−
+/−


M0004-



p9


F10


807B-
E22 P4
+(+)
+
p4, p9,
45
no RU3
43

11.55

low
++
+
−
−


M0004-



(p8)






RU4


H03


807B-
E22 P9,
(+)
+
p4, p9
23
no RU3
4

1.00

0.27
++
+++
+++
+++


M0009-
E24 P3


C03


807B-
E22 P9,
++
−
p4, p9
34
234
28

no RU3

10.6
+++
+++
+
+


M0009-
E24 P3


F06


807B-
E24
+++
+
(p3, p9),
27
23
26




++
++
+/−
+/−


M0013-



p4, p8


A12


807B-
E22&E23
(+)
+
p4, p8
1
2
1

43.64
1.05

+++
+++
+++
+++


M0079-
P9, E24


D10


807B-
E24
1n.d
+
(p9)
15
16
19

no RU3


+
++
+
+


M0081-


F12


807B-
E24
(+)
+
(p7)
9
18
7

no RU3


+++
+++
+++
+++


M0081-


H03


807B-
E24
−
+
no
12
64
25

no RU3


−
+
−
−


M0083-


E11


807A-
E5
+
+
P4
9
7
5




+++
+++
+++
+++


M0028-


B02


807A-
E5
(+)









+++
+++
1n.d
1n.d


M0026-


F05


807A-
E5
+









+++
+++
1n.d
1n.d


M0027-


E11





1n.d.: Not done


2IHC: tissue from human AD cortex used. 1 μg/ml of appropriate hIgG added


3no RU: no binding observed


4low RU: RUs are low, affinity was not measured


5VLDL ELISA: human VLDL coated 0.0003-100 μg/ml of appropriate hIgG tested for binding


6ELISA: human, mouse or primate CTD coated, 0.0003-100 μg/ml of appropriate hIgG tested for binding













TABLE 23







Binding of germ line corrected IgG to CTD and tissue













Affinity







(Biacore
VLDL
IHC3
ELISA4
In vitro phagocytosis















Isolate name
Peptide
(nM))
ELISA2
(tissue)
hCTD
mCTD
pCTD
EC50 ± SEM (ng/ml)


















807A-M0028-B02
P4
10.8
+++
++
+++
+++
+++
34 +/− 15


807A-M0028-B02.1
n.d.1
13
++
++
+++
+++
++
67 +/− 38


807A-M0028-B02.2
n.d.1

+++
++
+
+
+
27 +/− 7 


807B-M0004-H03
p4, p9, (p8)
12.5
++
++
+
−
+
53 +/− 25


807B-M0004-H03.1
n.d.1
17.7
−
+++
+++
−
++
22 +/− 16


807B-M0009-F06
p4, p9
14.8
+++
++
+++
+
++
23 +/− 9 


807B-M0009-F06.1
n.d.1
very
−
+
−
−
−
42 +/− 17




low




binding


807B-M0004-A03
(p1, p6) p4,
12.2
++
++
+++
+
++
205 +/− 81 



p8, p9


807B-M0004-A03.1
n.d.1
24.4
−
+3*
+++
+
+
78 +/− 43


807B-M0079-D10
p4, p8
n.d.1
+++
++
+++
+++
+++
  7 +/− 1.4


807B-M0079-D10.1
n.d.1
n.d.1
++
++
+++
+++
+++
19 +/− 15





1n.d.: Not done.


2VLDL ELISA: human VLDL is coated 0.0003-100 μg/ml of appropriate hIgG is tested for binding


3IHC: Brain tissue sections from APP/PS1 mouse was used. 1.5-1.8 μg/ml of appropriate hIgG is added,


3*reactivity to mCTD lower than to hCTD


4CTD ELISA: human, mouse or primate CTD is coated, 0.0003-100 μg/ml of appropriate hIgG is tested for binding













TABLE 24







807B-M0004-A03 = 39 clones selected for Fab production
















AminoAc
1(WT = S)
2(WT = I)
3(WT = A)
4(WT = A)
5(WT = A)
6(WT = G)
7(WT = T)
8(WT = D)
9(WT = Y)



















A


88%
65%
60%






D




9%


98%
2%


E



7%



2%


G
2%

5%
2%

84%


H






2%

21%


I

98%




2%


K






5%


L








2%


M

2%


N



5%


10%


P




5%


Q


2%


R
5%




16%
2%

5%


S
91%

5%
14%
19%


T
2%


2%
7%

79%


V



5%


Y








70%
















TABLE 25







807B-M0004-H03 = 54 clones selected for Fab production: part 1


















1(WT =











AminoAc
E)
2(WT = G)
3(WT = S)
4(WT = A)
5(WT = G)
6(WT = V)
7(WT = V)
8(WT = K)
9(WT = G)
10(WT = P)




















A
2%
2%
5%
82%
9%
5%


4%
5%


D
4%





2%


E
95%



2%
4%


F





4%
4%

2%


G

73%

2%
46%



73%


H









2%


I






7%
7%


K





2%

84%


L


5%


9%
9%


5%


M


2%


4%
2%


N







4%


P


5%




2%

77%


Q







2%

2%


R

18%
2%
9%
38%
4%

2%
11%
5%


S

4%
75%






4%


T





2%


V

4%

7%
4%
66%
77%


W


4%





9%


Y


Stop


2%
















TABLE 26







807B-M0004-H03 = 54 clones selected for Fab production: part 2


















11(WT =
12(WT =










AminoAc
A)
R)
13(WT = Y)
14(WT = Y)
15(WT = Y)
16(WT = Y)
17(WT = Y)
18(WT = M)
19(WT = D)
20(WT = V)




















A
73%







4%
4%


C


2%


D
2%





2%

77%


E






2%

4%


F


2%
4%

5%



5%


G
5%
2%






2%
2%


H


7%

7%
2%


4%


I







5%

7%


K

4%


L
2%
2%





2%

9%


M







93%


N


2%
4%
4%

4%

5%


P
4%
2%







2%


Q

4%




5%


R
2%
83%


S
5%



4%

5%

2%


T
5%


V
4%







2%
71%


Y

4%
88%
92%
85%
93%
82%

2%
















TABLE 27







807B-M0079-D10 = 33 clones selected for Fab production











AminoAc
1(WT = G)
2(WT = L)
3(WT = Y)
4(WT = R)














A
11%

6%



D
3%


F


9%


G
69%


3%


H


11%
9%


I

3%


K



3%


L

86%
3%
17%


M

6%


N


6%


P



3%


Q

3%

9%


R



43%


S
14%

6%
3%


T
3%


V

3%

3%


W



3%


Y


57%
6%


Stop


2%
















TABLE 28







807A-M0028-B02-CTD = 60 clones selected for Fab production













AminoAc
1(WT = S)
2(WT = V)
3(WT = L)
4(WT = L)
5(WT = D)
6(WT = Y)
















A
18%
2%


2%
2%


D

2%


92%


E




2%
2%


F
2%


19%

3%


G
19%
3%


H


3%
23%
2%
11%


I

23% 
2%
2%

2%


K





16%


L

2%
87%
52%

2%


M


3%


N

2%



11%


P
2%

2%
3%

3%


Q


2%


3%


R



2%

3%


S
57%




8%


T
2%
2%
2%


3%


V

66.50%   


Y
2%



2%
29%
















TABLE 29







807A-M0028-B02-fibrils = 12 clones selected for Fab production














1(WT =
2(WT =
3(WT =

5(WT =
6(WT =


AminoAc
S)
V)
L)
4(WT = L)
D)
Y)
















A
33%







D




100%


F



25%


G


H


8%
17%

17%


I


K

8%


L


92%
58%


N





17%


Q





8%


R





8%


S
67%




17%


V

92%


Y





33%
















TABLE 30







807B-M0009-F06 = 24 clones selected for Fab production


















1(WT =











AminoAc
V)
2(WT = G)
3(WT = M)
4(WT = S)
5(WT = T)
6(WT = Y)
7(WT = A)
8(WT = F)
9(WT = D)
10(WT = I)




















A

8%

8%


24%





D








96%


E








4%


F



4%



96%

4%


G

92%




60%


H




4%


I


20%






56%


K


4%






14%


L


12%
4%



4%

16%


M


60%






4%


N




12%


P



8%


S



60%
8%

16%


T



12%
76%




8%


V
100%

4%






8%


Y



4%

100%
















TABLE 31







Biacore analysis of 807B-M0004-A03, original clone and variants













BC
koff
kon
KD
Ranking














Clone
HV-CDR3
nM
1/s
1/Ms
nM
koff
KD

















*807B-M0004-
SIAAAGTDY
234
0.0294
3.18E+05
9.26E−08
27
13


A03/WT


807B-M0004-
SIAAAGTDY
232
0.0275
2.54E+05
1.08E−07
23
16


A03/WT


807B-M0004-
SIAADGIDY
181
0.0341
ND
3.07E+03
33
41


A03/M0117-A04


807B-M0004-
SIAATRTDY
239
0.0215
1.47E+05
1.46E−07
10
28


A03/M0117-A12


807B-M0004-
SIAAARTEY
192
0.0262
3.29E+04
7.97E−07
21
36


A03/M0117-B03


807B-M0004-
SIAPSGTDY
210
0.132
6.22E−04
212    
43
40


A03/M0117-B04


807B-M0004-
SIAPAGTDH
224
0.0297
2.45E+05
1.21E−07
28
22


A03/M0117-B05


807B-M0004-
SIAEAGTDY
245
0.0464
3.02E+05
1.54E−07
35
29


A03/M0117-B11


807B-M0004-
SIAVAGTDY
193
0.0902
1.25 
0.072
41
38


A03/M0117-C04


807B-M0004-
SIAGAGNDY
186
0.0306
2.40E+05
1.28E−07
30
23


A03/M0117-C07


807B-M0004-
SIAAAGTDH
280
0.0188
2.53E+05
7.44E−08
6
10


A03/M0117-C09


807B-M0004-
SIGAAGTDY
263
0.0767
0.0967
0.788
39
39


A03/M0117-C11


807B-M0004-
SIAASGTDY
134
0.0284
3.95E+05
7.22E−08
26
8


A03/M0117-C12


*807B-M0004-
SIAAARTDY
199
0.0128
9.45E+04
1.34E−07
3
25


A03/M0117-D03


807B-M0004-
SIQAAGTDH
123
0.0247
1.96E−05
4.21E+03
15
42


A03/M0117-E06


807B-M0004-
SIASPGTDY
228
0.0266
1.95E+05
1.37E−07
22
27


A03/M0117-E12


*807B-M0004-
SIASAGTDH
290
0.0139
2.22E+05
6.13E−08
4
4


A03/M0117-F05


807B-M0004-
GISTSGTDD
284
0.0243
2.08E+05
1.15E−07
14
19


A03/M0117-F11


807B-M0004-
SIAVAGTDY
235
0.0906
1.82E−05
4.96E+03
42
43


A03/M0117-F12


*807B-M0004-
SIASARTDS
256
0.0103
1.66E+05
6.07E−08
1
3


A03/M0117-G01


807B-M0004-
SIAAPGTDY
220
0.011
1.92E+04
4.84E−07
2
35


A03/M0117-G03


807B-M0004-
RIAASGTDY
92
0.049
4.42E+05
1.20E−07
36
21


A03/M0117-G04


807B-M0004-
SIAATGKDH
280
0.0815
9.94E+04
8.24E−07
40
37


A03/M0117-G05


807B-M0004-
SIAAAGSDS
471
0.0283
1.35E+05
2.23E−07
25
33


A03/M0117-G07


807B-M0004-
SIGASRTDY
336
0.0502
3.42E+05
1.68E−07
37
31


A03/M0117-H06


807B-M0004-
SIASAGTDL
217
0.0205
2.87E+05
6.97E−08
8
6


A03/M0117-H11


807B-M0004-
SIAAAGNDY
167
0.0254
3.51E+05
7.12E−08
17
7


A03/M0118-A03


*807B-M0004-
SIAADRTDY
252
0.0231
4.78E+05
4.70E−08
11
1


A03/M0118-B09


807B-M0004-
SIAESGTDY
203
0.0298
3.34E+05
9.06E−08
29
12


A03/M0118-B11


807B-M0004-
SIASSGTDH
213
0.0205
3.64E+05
5.47E−08
9
2


A03/M0118-C04


807B-M0004-
RMAAAGTDY
225
0.0235
9.81E+04
2.42E−07
12
34


A03/M0118-D02


807B-M0004-
SIAAAGKDY
281
0.0306
2.90E+05
1.33E−07
31
24


A03/M0118-D03


807B-M0004-
SIAATGTDI
263
0.028
2.54E+05
1.19E−07
24
20


A03/M0118-D07


807B-M0004-
SIAAAGNDH
179
0.0236
3.19E+05
7.41E−08
13
9


A03/M0118-E10


807B-M0004-
SIASAGTDY
267
0.0312
2.52E+05
1.55E−07
32
30


A03/M0118-E12


*807B-M0004-
SIAASRTDY
230
0.0158
2.36E+05
6.56E−08
5
5


A03/M0118-F03


807B-M0004-
SIAAAGTDH
372
0.0194
2.38E+05
7.97E−08
7
11


A03/M0118-F06


807B-M0004-
SIAEAGTDY
245
0.0456
2.92E+05
1.78E−07
34
32


A03/M0118-F09


807B-M0004-
SISAAGTDY
278
0.0249
1.78E+05
1.37E−07
16
26


A03/M0118-F12


807B-M0004-
SIAADGTDY
208
0.0669
6.16E+05
1.14E−07
38
18


A03/M0118-G03


807B-M0004-
TIAAAGTDY
267
0.0259
2.42E+05
1.13E−07
20
17


A03/M0118-G05


807B-M0004-
SIAAAGHDH
336
0.0256
2.70E+05
1.00E−07
18
14


A03/M0118-G08


807B-M0004-
SIAAAGNDY
276
0.0258
2.69E+05
1.00E−07
19
15


A03/M0118-H01





*= selected clone


BC = Biacore













TABLE 32







Biacore analysis of 807B-M0004-H03, original clone and variants













Biacore
koff
kon
KD
Ranking














Clone
HV-CDR3
nM
1/s
1/Ms
nM
koff
KD

















*807B-M0004-H03/WT
EGSAGVVKGPARYYYYYMDV
703
1.18E−03
4.37E+03
2.56E−07
1
16


807B-M0004-H03/M0119-A07
ERSAGVLKGPAWYYYYYMDV
262
5.94E−03
1.48E+04
1.67E−07
36
7


807B-M0004-H03/M0119-A08
EGSAAFVKGPARYYYYDMDI
375
2.66E−01
1.24E+04
4.64E−07
55
30


*807B-M0004-H03/M0119-B05
EGSSGVVKGPARYYYYYMDA
371
1.53E−03
6.03E+03
1.59E−07
4
5


807B-M0004-H03/M0119-B06
EGSVGAVKGRARYYYYYMNV
729
2.62E−03
3.45E+03
4.00E−07
16
26


807B-M0004-H03/M0119-B07
EGSAGVFKGPARYYYYYMDV
486
7.20E−03
1.36E+04
3.98E−07
40
24


*807B-M0004-H03/M0119-C05
ERSVAVFKARPRHYYYYMDV
696
1.69E−03
1.09E+04
1.46E−07
5
3


807B-M0004-H03/M0119-C08
EGSAGVDIGPARYYYYYMNV
453
7.24E−03
1.46E+04
4.72E−07
41
32


807B-M0004-H03/M0119-C11
EGSAAVVKAPAKYYYYYMEV
347
7.39E−03
1.47E+04
4.70E−07
43
31


807B-M0004-H03/M0119-E01
EGSVGVVKGPARYYHYQIDV
541
3.03E−03
7.69E+03
3.29E−07
19
21


807B-M0004-H03/M0119-E04
ESSARVVKGLARYYNYYMHV
484
5.34E−03
5.43E+03
6.00E−07
31
38


807B-M0004-H03/M0119-E07
ERPSRVVKGPTRYYYYYMDV
577
5.51E−03
4.33E−03
7.70E−01
32
52


807B-M0004-H03/M0119-E11
EVSARVVKCPARYYYYYMDV
614
9.69E−03
7.74E−06
1.19E+03
45
54


807B-M0004-H03/M0119-F01
EGSAGVIKGPARYYYFYMGV
389
1.85E−01
1.38E+04
7.14E−07
54
41


*807B-M0004-H03/M0119-F04
EGSARVIKGPARYYYYEMDV
675
3.99E−03
2.68E+04
1.44E−07
29
2


807B-M0004-H03/M0119-F05
ERSVGVVIGHARYFYYYMDV
472
1.72E−03
8.05E+03
1.99E−07
6
8


807B-M0004-H03/M0119-F09
EGPAGVVKGRARYYSYNMSV
246
1.13E−01
8.96E+05
1.10E−07
52
1


807B-M0004-H03/M0119-F10
ESSARVVNGPAWYYYYYMDA
261
5.71E−03
8.52E+03
6.47E−07
35
39


807B-M0004-H03/M0119-F11
EGSSRAVKGAPRYYYYYMDV
1027
3.41E−03
1.52E+03
1.68E−06
24
45


807B-M0004-H03/M0119-F12
EVSGGVVKGPARYYYYYMAL
862
2.57E−02
6.16E+03
3.93E−06
48
47


807B-M0004-H03/M0119-G08
EGqARRVKGQARYYYYYMDV
270
3.33E−03
3.29E+03
6.81E−07
23
40


807B-M0004-H03/M0119-G10
EGSAGLVKGPARYYYYYMDV
514
1.76E−03
4.94E+03
2.95E−07
7
19


807B-M0004-H03/M0119-G12
ERSAGVVKGPSRNYYYYMDV
728
3.73E−03
1.56E+04
2.36E−07
27
14


807B-M0004-H03/M0119-H01
EGSARRVKRPGRYYYYQMDV
912
1.06E−02
3.66E+04
2.84E−07
46
18


807B-M0004-H03/M0119-H03
EGSARMLKGPARCYYYYMDV
315
7.36E−03
1.55E+04
4.38E−07
42
28


807B-M0004-H03/M0119-H08
EGMAGVVKFPARHNYHYMDV
881
4.58E−02
8.86E+04
5.76E−07
49
37


*807B-M0004-H03/M0119-H09
DGSARVVKGPRRYYYYYIDV
351
1.40E−03
7.92E+03
1.50E−07
2
4


807B-M0004-H03/M0119-H11
ERPAGLVKGPARYYSYYMDV
1205
2.21E−03
6.55E+03
3.11E−07
12
20


807B-M0004-H03/M0120-A03
EGSARMVKGAARYYYYYMDV
1109
1.62E−02
6.25E+04
2.50E−07
47
15


807B-M0004-H03/M0120-A07
EGSAGTIKWLVRYYNFYMDV
707
3.24E−03
6.77E+03
4.48E−07
21
29


*807B-M0004-H03/M0120-B05
EGSARVVKGPARYFYYYMDL
740
1.40E−03
4.70E+03
2.34E−07
3
13


807B-M0004-H03/M0120-B06
EGSARVVKGPDRYYYYYMAP
309
5.62E−03
8.84E−03
4.82E−01
33
51


807B-M0004-H03/M0120-B09
EGSAGKVIGPAPHYYYYMDV
868
6.90E−03
2.05E+04
3.34E−07
39
22


807B-M0004-H03/M0120-B11
EGRARVLKGLARYYHYYMDF
1023
3.01E−03
3.50E+02
5.42E−06
18
49


807B-M0004-H03/M0120-C02
EGSARFVKGPARYYYYYMDI
779
3.74E−03
5.68E+02
4.10E−06
28
48


807B-M0004-H03/M0120-C06
EGSSRLVQWPARYYYYSMDV
747
3.67E−03
5.96E+03
5.18E−07
26
35


807B-M0004-H03/M0120-C07
ERSAGVMKGPTLYYYYYMDV
641
8.85E−03
6.51E+03
1.17E−06
44
44


807B-M0004-H03/M0120-C12
EGSAGVVNRSSRYNYYYLDV
959
4.94E−02
ND
ND
50
—


807B-M0004-H03/M0120-D04
EGSSVEVKGPARYYHYYMDV
652
1.85E−03
6.71E+03
2.07E−07
10
9


807B-M0004-H03/M0120-D05
EGSAGVVKGPTRYYYYSMDV
921
2.28E−03
3.84E+03
4.81E−07
15
33


807B-M0004-H03/M0120-E02
EGSAVVVKRSARYYYYYMNF
827
6.72E−03
6.74E+03
8.20E−07
38
42


807B-M0004-H03/M0120-E04
ERSARLLKGPLRYYYYYMDV
817
1.86E−03
2.35E+03
3.70E−07
11
23


807B-M0004-H03/M0120-E06
ERAARAVKGPSRYYYYYMHV
1110
1.15E−01
3.93E−03
3.76E+00
53
53


807B-M0004-H03/M0120-F03
EGLAGVVKRPARFYYYYMDV
873
3.26E−03
1.09E+04
2.80E−07
22
17


807B-M0004-H03/M0120-F04
EGSARVVIWPAQYYYYYMDF
434
3.53E−03
5.48E+03
4.89E−07
25
34


807B-M0004-H03/M0120-F06
EGSARVVKGPARYYYYSMVV
850
2.94E−03
4.44E+03
4.17E−07
17
27


807B-M0004-H03/M0120-F07
ERSAAVVKWPVRYYYYYMDL
611
4.92E−03
1.26E+03
2.41E−06
30
46


807B-M0004-H03/M0120-G02
EGWAALVKGPGRYYYYQMYV
749
3.19E−01
4.67E+02
2.80E−05
56
50


807B-M0004-H03/M0120-G03
EGSAGVLKGPAKYYYYYMDI
755
1.77E−03
6.24E+03
2.33E−07
8
12


807B-M0004-H03/M0120-G04
EGSARVVKGPARYYYYYMDV
956
1.81E−03
1.09E+04
1.64E−07
9
6


807B-M0004-H03/M0120-G10
EGSAGVVKGPARHYYYYMDI
755
2.28E−03
9.65E+03
2.30E−07
14
11


807B-M0004-H03/M0120-G12
DGLAEEVKGPAQYYYYYIDG
782
7.80E−02
7.72E+04
1.02E−06
51
43


807B-M0004-H03/M0120-H03
EAAAGVVKGPARYYYFNMEV
1114
2.27E−03
4.87E+03
3.99E−07
13
25


807B-M0004-H03/M0120-H04
EGAARAVRRPAGYYHYYMDL
916
3.22E−03
1.46E+04
2.16E−07
20
10


807B-M0004-H03/M0120-H06
QGWAGVVKWPARYYYYYMDV
3
5.71E−03
ND
ND
34
—


807B-M0004-H03/M0120-H10
EGLAGVIPRAARYYYYYMDL
800
6.12E−03
1.06E+04
5.45E−07
37
36





*= selected clones













TABLE 33







Biacore analysis of 807B-M0009-F06, original clone and variants













Biacore
koff
kon
KD
Ranking














Clone
HV-CDR3
nM
1/s
1/Ms
nM
koff
KD

















807B-M0009-F06/WT
VGMSTYAFDI
243
9.70E−02
0.043 
2.26
23
25


807B-M0009-
VGMSTYGLEI
471
2.79E−02
2.14E+04
1.26E−06
17
18


F06/M0127-A01


*807B-M0009-
VGMSTYGFDK
418
8.83E−03
5.62E+04
1.56E−07
2
8


F06/M0127-B07


807B-M0009-
VGMTTYAFDV
390
8.59E−02
0.0434
1.85
20
22


F06/M0127-B08


*807B-M0009-
VGISTYGFDL
78
1.53E−02
1.74E+05
8.68E−08
10
2


F06/M0127-C10


807B-M0009-
VGISTYGFDI
324
1.45E−02
5.56E+04
2.57E−07
7
15


F06/M0127-D01


*807B-M0009-
VGMATYGFDI
346
9.45E−03
6.97E+04
1.34E−07
3
5


F06/M0127-D05


807B-M0009-
VGISTYGFDV
240
1.49E−02
1.09E+05
1.35E−07
9
6


F06/M0127-E03


807B-M0009-
VGMSTYGFDI
289
1.05E−02
7.43E+04
1.40E−07
5
7


F06/M0127-E10


807B-M0009-
VGIPTYSFDI
146
2.24E−02
2.61E+04
8.57E−07
14
17


F06/M0127-E11


807B-M0009-
VGLATYSFDL
203
1.02E−01
0.0628
1.62
25
20


F06/M0127-F07


*807B-M0009-
VGMYNYGFDI
221
1.02E−02
1.09E+05
9.38E−08
4
3


F06/M0127-F09


807B-M0009-
VGMSTYSFDT
271
1.01E−01
0.0453
2.23
24
24


F06/M0127-F11


807B-M0009-
VGVSTYGFDI
39
1.59E−02
1.47E+05
1.08E−07
11
4


F06/M0127-G02


807B-M0009-
VGMFTYAFDT
201
7.87E−02
0.0435
1.81
19
21


F06/M0127-H04


807B-M0009-
VGKSTYGFDI
305
1.79E−02
8.37E+04
2.14E−07
12
13


F06/M0127-H05


807B-M0009-
VAMTTYGFDL
253
2.41E−02
3.70E+04
6.52E−07
16
16


F06/M0128-C01


807B-M0009-
VGISSYGFDI
316
1.46E−02
7.76E+04
1.88E−07
8
11


F06/M0128-D09


807B-M0009-
VAMSNYGFDL
159
2.38E−02
1.13E+05
2.10E−07
15
12


F06/M0128-D12


807B-M0009-
VGMTHYAFDI
148
1.98E−02
7.91E+04
2.50E−07
13
14


F06/M0128-F02


807B-M0009-
VGMLTYAFDI
120
7.71E−02
0.0765
1.01
18
19


F06/M0128-F08


807B-M0009-
VGLPSYSFDI
115
8.98E−02
1.24E+05
1.72E−07
22
10


F06/M0128-G09


*807B-M0009-
VGMSNYGFDF
116
7.78E−03
1.62E+05
4.79E−08
1
1


F06/M0128-H01


807B-M0009-
VGMSTYAFDM
211
8.98E−02
0.048 
1.87
21
23


F06/M0128-H07


807B-M0009-
VGLSTYGFDI
295
1.31E−02
8.09E+04
1.62E−07
6
9


F06/M0128-H11





*= selected clones













TABLE 34







Biacore screening of 807B-M0079-D10, original clone and variants













koff



Clone
HV-CDR3
1/s







*807B-M0079-D10/WT
GLYR
6.02E−03



807B-M0079-D10/M0121-A01
GLHR
1.29E−02



807B-M0079-D10/M0121-A02
GLYG
1.95E−02



807B-M0079-D10/M0121-A06
GLYH
1.47E−02



807B-M0079-D10/M0121-A08
GLHL
2.14E−02



807B-M0079-D10/M0121-A11
GIYR
1.43E−02



807B-M0079-D10/M0121-A12
ALAR
1.65E−02



807B-M0079-D10/M0121-B04
GLFR
1.07E−02



807B-M0079-D10/M0121-B05
SLYQ
2.31E−02



807B-M0079-D10/M0121-B12
GLLL
1.71E−02



807B-M0079-D10/M0121-C01
GQYR
1.93E−02



807B-M0079-D10/M0121-C03
GLAR
2.24E−02



807B-M0079-D10/M0121-D01
GLYQ
1.34E−02



807B-M0079-D10/M0121-D05
GLYP
5.93E−03



807B-M0079-D10/M0121-D06
GMYR
1.62E−02



807B-M0079-D10/M0121-E02
ALYS
5.34E−03



807B-M0079-D10/M0121-F02
GLSR
8.02E−03



807B-M0079-D10/M0121-F05
SLYL
1.84E−02



807B-M0079-D10/M0121-F06
GLYL
1.11E−02



807B-M0079-D10/M0121-F11
GMYV
5.92E−03



807B-M0079-D10/M0121-G03
SLYR
1.22E−02



807B-M0079-D10/M0121-G10
ALYR
1.03E−02



807B-M0079-D10/M0121-H04
SLYH
2.03E−02



807B-M0079-D10/M0121-H05
GLYY
1.70E−02



807B-M0079-D10/M0122-A01
DLYR
1.65E−02



807B-M0079-D10/M0122-B03
TLHR
2.58E−02



807B-M0079-D10/M0122-D01
GLHH
2.11E−02



807B-M0079-D10/M0122-D03
GLNR
1.29E−02



807B-M0079-D10/M0122-D05
GLSQ
1.43E−02



807B-M0079-D10/M0122-E06
GLqR
2.59E−02



807B-M0079-D10/M0122-F09
GLFY
8.76E−03



807B-M0079-D10/M0122-F11
GLNL
1.21E−02



807B-M0079-D10/M0122-G07
SLFK
2.17E−02



807B-M0079-D10/M0122-G12
ALYW
6.11E−03



807B-M0079-D10/M0122-H11
GVYL
1.32E−02







*= selected clones













TABLE 35







Biacore screening of 807A-M0028-B02, original clone and variants













BC
koff
kon
KD
Ranking














Clone
HV-CDR3
nM
1/s
1/Ms
nM
koff
KD

















*807A-M0028-B02/WT
SVLLDY
592
1.48E−02
1.73E+04
869
72
61


807A-M0028-B02/M0123-A04
SVQLYP
120
5.05E−02
0.0776
660000000
74
73


807A-M0028-B02/M0123-A05
SVLHDK
958
8.85E−03
1.16E+04
768
18
57


807A-M0028-B02/M0123-A06
FALLDY
247
7.82E−03
2.20E+04
358
13
28


807A-M0028-B02/M0123-A07
SVLFDK
548
6.97E−03
1.96E+04
358
10
27


807A-M0028-B02/M0123-A09
TLLLDs
345
1.47E−02
5.85E−03
2530000000
71
74


807A-M0028-B02/M0123-A10
GVLLDL
385
9.74E−03
2.13E+04
462
27
40


807A-M0028-B02/M0123-A11
SVLFDY
398
1.30E−02
1.29E+04
1020
66
67


807A-M0028-B02/M0123-A12
SILFDY
449
1.02E−02
1.36E+04
757
33
56


807A-M0028-B02/M0123-B01
SVLLDQ
711
9.49E−03
8.59E+03
1110
25
70


807A-M0028-B02/M0123-B03
SNLHDQ
199
1.21E−02
1.12E+04
1080
55
69


807A-M0028-B02/M0123-B06
AILLNY
207
7.43E−03
2.89E+04
257
12
15


807A-M0028-B02/M0123-B08
AVLLDH
471
1.03E−02
1.38E+04
745
34
54


807A-M0028-B02/M0123-B10
AVMHDK
858
3.64E−03
1.80E+04
202
1
8


807A-M0028-B02/M0123-C07
SVLFDS
706
1.25E−02
1.32E+04
945
61
63


807A-M0028-B02/M0123-C11
GVLLDI
345
9.78E−03
3.11E+04
315
29
20


*807A-M0028-B02/M0123-D01
GVLLDK
578
5.62E−03
2.65E+04
212
6
9


807A-M0028-B02/M0123-D03
SVLLDN
710
1.12E−02
1.09E+04
1030
45
68


807A-M0028-B02/M0123-D04
SVLHDY
592
1.41E−02
1.22E+04
1160
70
71


807A-M0028-B02/M0123-D06
SVLFDR
510
9.07E−03
2.13E+04
427
20
35


807A-M0028-B02/M0123-D08
SVLLDK
1013
9.26E−03
1.08E+04
862
23
60


807A-M0028-B02/M0123-E05
GGLLDY
884
1.06E−02
1.10E+04
976
37
65


807A-M0028-B02/M0123-E12
SVMFDY
646
1.13E−02
1.14E+04
1000
46
66


807A-M0028-B02/M0123-F01
SILHDY
1049
1.21E−02
7.34E+03
1660
56
72


*807A-M0028-B02/M0123-F04
GILHDY
718
4.82E−03
2.07E+04
233
5
13


807A-M0028-B02/M0123-F11
SVIFDY
522
1.26E−02
1.72E+04
739
62
53


807A-M0028-B02/M0123-F12
SILFDN
737
9.08E−03
1.45E+04
630
21
49


807A-M0028-B02/M0123-G02
AILLDY
190
1.55E−02
2.09E+04
747
73
55


807A-M0028-B02/M0123-G03
AILLDH
394
1.25E−02
1.76E+04
717
60
52


807A-M0028-B02/M0123-G12
SILFDT
628
8.17E−03
2.01E+04
409
15
34


807A-M0028-B02/M0123-H02
AVLLDY
276
1.11E−02
2.77E+04
404
42
33


807A-M0028-B02/M0123-H09
SVLPDN
685
8.78E−03
2.04E+04
434
16
36


807A-M0028-B02/M0123-H10
GILLDK
591
6.36E−03
2.85E+04
224
8
10


807A-M0028-B02/M0123-H11
SVLFDN
619
1.15E−02
1.95E+04
596
48
47


807A-M0028-B02/M0124-A01
SVLLDS
738
6.01E−03
1.75E+04
344
7
23


807A-M0028-B02/M0124-A10
SDLRAE
815
9.75E−03
1.15E+04
855
28
59


807A-M0028-B02/M0124-A11
GVLLDY
452
8.85E−03
3.47E+04
257
17
16


*807A-M0028-B02/M0124-B02
GVLHDY
649
4.40E−03
2.66E+04
166
3
5


807A-M0028-B02/M0124-B03
SVLLDR
201
1.25E−02
4.88E+04
258
59
17


807A-M0028-B02/M0124-B08
SILHDK
590
6.37E−03
3.20E+04
200
9
7


*807A-M0028-B02/M0124-B11
SILFDK
389
4.49E−03
3.82E+04
118
4
2


807A-M0028-B02/M0124-C01
SILLDH
517
1.17E−02
2.00E+04
590
49
46


807A-M0028-B02/M0124-C02
SVPIDH
243
1.24E−02
3.63E+04
344
58
24


807A-M0028-B02/M0124-C03
PVLLHF
269
8.87E−03
3.87E+04
231
19
11


807A-M0028-B02/M0124-C04
GVLLEP
509
9.99E−03
2.05E+04
494
31
41


*807A-M0028-B02/M0124-C05
GVLFDN
230
3.93E−03
7.01E+04
56
2
1


807A-M0028-B02/M0124-C06
AJLLDK
272
8.08E−03
5.04E+04
162
14
4


807A-M0028-B02/M0124-D02
STLLDH
445
1.11E−02
2.07E+04
541
43
43


807A-M0028-B02/M0124-D06
SIHLDY
470
1.23E−02
1.40E+04
888
57
62


807A-M0028-B02/M0124-D08
SVTLDA
446
1.20E−02
2.23E+04
543
53
45


807A-M0028-B02/M0124-D09
SVLHDF
378
1.30E−02
2.87E+04
459
65
39


807A-M0028-B02/M0124-D10
SVLHDS
390
1.39E−02
2.22E+04
634
69
51


807A-M0028-B02/M0124-D12
GGLLDK
853
7.07E−03
2.16E+04
328
11
22


807A-M0028-B02/M0124-E02
AVLLDT
459
1.04E−02
2.74E+04
383
35
30


807A-M0028-B02/M0124-E03
AVLHDY
490
1.00E−02
2.21E+04
456
32
38


807A-M0028-B02/M0124-E04
SVLHDQ
272
1.21E−02
3.06E+04
399
54
32


807A-M0028-B02/M0124-E10
GVLLDN
493
9.68E−03
2.74E+04
357
26
26


807A-M0028-B02/M0124-F03
SVLLDH
513
9.24E−03
2.87E+04
325
22
21


807A-M0028-B02/M0124-F05
AVLHDS
211
1.19E−02
2.66E+04
451
52
37


807A-M0028-B02/M0124-G03
YVHPDY
655
1.32E−02
1.37E+04
971
68
64


807A-M0028-B02/M0124-G07
SVLHDH
516
9.81E−03
2.61E+04
380
30
29


807A-M0028-B02/M0124-G10
AVLLDN
316
1.08E−02
3.87E+04
282
38
18


807A-M0028-B02/M0125-C03
SVLLDR
473
1.18E−02
3.12E+04
385
50
31


807A-M0028-B02/M0125-D03
SVLFDY
295
1.11E−02
4.41E+04
254
40
14


807A-M0028-B02/M0125-D06
SVHLDY
161
1.12E−02
8.30E+04
136
44
3


807A-M0028-B02/M0125-D09
AVLHDS
639
1.06E−02
1.97E+04
542
36
44


807A-M0028-B02/M0125-F07
SVLLDQ
607
1.29E−02
2.06E+04
631
64
50


807A-M0028-B02/M0125-F11
SVLFDS
621
1.18E−02
2.39E+04
498
51
42


807A-M0028-B02/M0125-G02
SVLLDH
511
1.32E−02
2.21E+04
607
67
48


807A-M0028-B02/M0126-C09
AVLLDY
329
1.10E−02
3.18E+04
350
39
25


807A-M0028-B02/M0126-E03
AVLLDN
257
1.11E−02
3.86E+04
290
41
19


807A-M0028-B02/M0126-F08
SILFDY
335
9.35E−03
4.07E+04
232
24
12


807A-M0028-B02/M0126-G03
SVLHDN
627
1.28E−02
1.53E+04
846
63
58


807A-M0028-B02/M0126-G07
AVLLDH
221
1.15E−02
5.41E+04
194
47
6





*= selected clones


BC = Biacore













TABLE 36







Detailed biacore analysis of 807B-M0004-A03, 807B-M0009-F06,


807A-M0028-B02 and variants











Clone name
kon (1/Ms)
koff (1/s)
KD (nM)
CDR3














807B-M004-A03/WT
1.82E+05
2.34E−02
128
SIAAAGTDY


807B-M004-A03/M0118-B09
3.12E+05
1.96E−02
63
SIAADRTDY


807B-M004-A03/M0117-G01
1.39E+05
7.72E−03
56
SIASARTDS


807B-M004-A03/M0117-D03
2.75E+04
8.36E−03
304
SIAAARTDY


807B-M004-A03/M0117-F05
1.89E+05
1.05E−02
56
SIASAGTDH


807B-M004-A03/M0118-F03
1.35E+05
1.16E−02
86
SIAASRTDY


807B-M0009F06/WT
1.31E+05
4.45E−02
340
VGMSTYAFDI


807B-M0009F06-M0128-H01
8.72E+04
7.99E−03
92
VGMSNYGFDF


807B-M0009F06-M0127-B07
1.00E+05
8.60E−03
86
VGMSTYGFDK


807B-M0009F06-M0127-D05
1.02E+05
9.39E−03
92
VGMATYGFDI


807B-M0009F06-M0127-F09
1.18E+05
1.10E−02
93
VGMYNYGFDI


807B-M0009F06-M0127-C10
1.13E+05
1.62E−02
144
VGISTYGFDL


807A-M0028-B02/WT
9.31E+04
1.58E−02
169
SVLLDY


807A-M0028-B02/M0124-C05
2.76E+04
5.12E−03
185
GVLFDN


807A-M0028-B02/M0124-B02
6.07E+04
6.77E−03
112
GVLHDY


807A-M0028-B02/M0124-B11
5.23E+04
5.72E−03
109
SILFDK


807A-M0028-B02/M0123-F04
5.07E+04
7.56E−03
149
GILHDY


807A-M0028-B02/M0123-D01
9.25E+04
7.85E−03
85
GVLLDK


807B-M0004-H03/WT
—
—
200
EGSAGVVKGP






ARYYYYYMDV


807B-M0004-H03/M0119-B05
—
—
—
EGSSGVVKGP






ARYYYYYMDA


807B-M0004-H03/M0119-C05
—
—
—
ERSVAVFKAR






PRHYYYYMDV


807B-M0004-H03/M0119-F04
—
—
—
EGSARVIKGP






ARYYYYEMDV


807B-M0004-H03/M0119-H09
—
—
—
DGSARVVKGP






RRYYYYYIDV


807B-M0004-H03/M0120-B05
—
—
—
EGSARVVKGP






ARYFYYYMDL
















TABLE 37







Immunohistochemistry of clones selected from Biacore screening









IHC














Clone 807B-M0004-A03




Original clone
+/−



807B-M0004-A03/M0117-D03
+



807B-M0004-A03/M0117-F05
+



807B-M0004-A03/M0117-G01
+/−



807B-M0004-A03/M0118-B09
+(+)



807B-M0004-A03/M0118-F03
+/−



Clone 807B-M0004-H03



Original clone
+/−



807B-M0004-H03/M0119-B05
++(+)



807B-M0004-H03/M0119-C05
++(+)



807B-M0004-H03/M0119-F04
++(+)



807B-M0004-H03/M0119-H09
++(+)



807B-M0004-H03/M0120-B05
++



Clone 807A-M0028-B02



Original clone
++



807B-M0028-B02/M0123-D01
++



807B-M0028-B02/M0123-F04
++



807B-M0028-B02/M0124-B02
++



807B-M0028-B02/M0124-B11
++



807B-M0028-B02/M0124-C05
++



Clone 807B-M0009-F06



Original clone
+/−



807B-M0009-F06/M0127-B07
+/−



807B-M0009-F06/M0127-C10
+



807B-M0009-F06/M0127-D05
−



807B-M0009-F06/M0127-F09
+/−



807B-M0009-F06/M0128-H01
+

















TABLE 38





Affinity matured clones of 807A-M0028-B02



















Initial Name
HV-CDR3
LV-CDR1
LV-CDR2
LV-CDR3





807A-M0028-B02/M0167-E01
SEQ ID NO: 207
SEQ ID NO: 211
SEQ ID NO: 240
SEQ ID NO: 262


807A-M0028-B02/M0167-E07
SEQ ID NO: 208
SEQ ID NO: 212
SEQ ID NO: 241
SEQ ID NO: 263


807A-M0028-B02/M0167-F07
SEQ ID NO: 209
SEQ ID NO: 213
SEQ ID NO: 34
SEQ ID NO: 35


807A-M0028-B02/M0167-F09
SEQ ID NO: 207
SEQ ID NO: 214
SEQ ID NO: 243
SEQ ID NO: 265


807A-M0028-B02/M0168-B11
SEQ ID NO: 209
SEQ ID NO: 215
SEQ ID NO: 244
SEQ ID NO: 266


807A-M0028-B02/M0168-C08
SEQ ID NO: 209
SEQ ID NO: 216
SEQ ID NO: 245
SEQ ID NO: 267


807A-M0028-B02/M0168-D10
SEQ ID NO: 210
SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 35


807A-M0028-B02/M0169-D08
SEQ ID NO: 208
SEQ ID NO: 218
SEQ ID NO: 34
SEQ ID NO: 268


807A-M0028-B02/M0169-F03
SEQ ID NO: 209
SEQ ID NO: 219
SEQ ID NO: 247
SEQ ID NO: 269


807A-M0028-B02/M0169-H04
SEQ ID NO: 208
SEQ ID NO: 220
SEQ ID NO: 248
SEQ ID NO: 270


807A-M0028-B02/M0169-H05
SEQ ID NO: 207
SEQ ID NO: 221
SEQ ID NO: 249
SEQ ID NO: 271


807A-M0028-B02/M0170-H08
SEQ ID NO: 207
SEQ ID NO: 222
SEQ ID NO: 250
SEQ ID NO: 272


807A-M0028-B02/M0171-A08
SEQ ID NO: 210
SEQ ID NO: 223
SEQ ID NO: 240
SEQ ID NO: 273


807A-M0028-B02/M0171-A09
SEQ ID NO: 209
SEQ ID NO: 224
SEQ ID NO: 251
SEQ ID NO: 274


807A-M0028-B02/M0171-A10
SEQ ID NO: 207
SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 35


807A-M0028-B02/M0171-C12
SEQ ID NO: 207
SEQ ID NO: 226
SEQ ID NO: 252
SEQ ID NO: 275


807A-M0028-B02/M0171-E03
SEQ ID NO: 209
SEQ ID NO: 218
SEQ ID NO: 34
SEQ ID NO: 268


807A-M0028-B02/M0171-G02
SEQ ID NO: 208
SEQ ID NO: 228
SEQ ID NO: 253
SEQ ID NO: 276


807A-M0028-B02/M0171-G09
SEQ ID NO: 209
SEQ ID NO: 229
SEQ ID NO: 254
SEQ ID NO: 277


807A-M0028-B02/M0172-A05
SEQ ID NO: 209
SEQ ID NO: 230
SEQ ID NO: 255
SEQ ID NO: 278


807A-M0028-B02/M0172-A08
SEQ ID NO: 208
SEQ ID NO: 231
SEQ ID NO: 256
SEQ ID NO: 279


807A-M0028-B02/M0172-B09
SEQ ID NO: 209
SEQ ID NO: 232
SEQ ID NO: 257
SEQ ID NO: 280


807A-M0028-B02/M0172-D05
SEQ ID NO: 209
SEQ ID NO: 233
SEQ ID NO: 257
SEQ ID NO: 281


807A-M0028-B02/M0172-D09
SEQ ID NO: 207
SEQ ID NO: 234
SEQ ID NO: 258
SEQ ID NO: 282


807A-M0028-B02/M0172-E06
SEQ ID NO: 209
SEQ ID NO: 235
SEQ ID NO: 251
SEQ ID NO: 283


807A-M0028-B02/M0172-F02
SEQ ID NO: 207
SEQ ID NO: 236
SEQ ID NO: 259
SEQ ID NO: 284


807A-M0028-B02/M0172-F07
SEQ ID NO: 210
SEQ ID NO: 213
SEQ ID NO: 34
SEQ ID NO: 35


807A-M0028-B02/M0172-F12
SEQ ID NO: 209
SEQ ID NO: 238
SEQ ID NO: 260
SEQ ID NO: 285


807A-M0028-B02/M0172-G08
SEQ ID NO: 209
SEQ ID NO: 239
SEQ ID NO: 261
SEQ ID NO: 286





Initial Name
HV-CDR1
HV-CDR2
LV-WholeAA
HV-WholeAA





807A-M0028-B02/M0167-E01
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 287
SEQ ID NO: 316


807A-M0028-B02/M0167-E07
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 288
SEQ ID NO: 317


807A-M0028-B02/M0167-F07
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 289
SEQ ID NO: 318


807A-M0028-B02/M0167-F09
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 290
SEQ ID NO: 316


807A-M0028-B02/M0168-B11
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 291
SEQ ID NO: 318


807A-M0028-B02/M0168-C08
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 292
SEQ ID NO: 318


807A-M0028-B02/M0168-D10
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 43
SEQ ID NO: 319


807A-M0028-B02/M0169-D08
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 294
SEQ ID NO: 317


807A-M0028-B02/M0169-F03
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 295
SEQ ID NO: 318


807A-M0028-B02/M0169-H04
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 296
SEQ ID NO: 317


807A-M0028-B02/M0169-H05
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 297
SEQ ID NO: 316


807A-M0028-B02/M0170-H08
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 298
SEQ ID NO: 316


807A-M0028-B02/M0171-A08
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 299
SEQ ID NO: 319


807A-M0028-B02/M0171-A09
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 300
SEQ ID NO: 318


807A-M0028-B02/M0171-A10
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 43
SEQ ID NO: 316


807A-M0028-B02/M0171-C12
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 302
SEQ ID NO: 316


807A-M0028-B02/M0171-E03
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 294
SEQ ID NO: 318


807A-M0028-B02/M0171-G02
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 304
SEQ ID NO: 317


807A-M0028-B02/M0171-G09
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 305
SEQ ID NO: 318


807A-M0028-B02/M0172-A05
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 306
SEQ ID NO: 318


807A-M0028-B02/M0172-A08
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 307
SEQ ID NO: 317


807A-M0028-B02/M0172-B09
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 308
SEQ ID NO: 318


807A-M0028-B02/M0172-D05
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 309
SEQ ID NO: 318


807A-M0028-B02/M0172-D09
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 310
SEQ ID NO: 316


807A-M0028-B02/M0172-E06
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 311
SEQ ID NO: 318


807A-M0028-B02/M0172-F02
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 312
SEQ ID NO: 316


807A-M0028-B02/M0172-F07
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 289
SEQ ID NO: 319


807A-M0028-B02/M0172-F12
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 314
SEQ ID NO: 318


807A-M0028-B02/M0172-G08
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 315
SEQ ID NO: 318
















TABLE 39





Affinity matured clones of 807B-M0004-A03



















Initial Name
HV-CDR3_1
LV-CDR1
LV-CDR2
LV-CDR3





807B-M0004-A03/M0148-E05
SEQ ID NO: 320
SEQ ID NO: 93
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0148-E08
SEQ ID NO: 320
SEQ ID NO: 325
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0149-D04
SEQ ID NO: 322
SEQ ID NO: 326
SEQ ID NO: 334
SEQ ID NO: 341


807B-M0004-A03/M0149-F02
SEQ ID NO: 322
SEQ ID NO: 93
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0149-G11
SEQ ID NO: 323
SEQ ID NO: 93
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0149-H07
SEQ ID NO: 322
SEQ ID NO: 326
SEQ ID NO: 334
SEQ ID NO: 341


807B-M0004-A03/M0149-H09
SEQ ID NO: 320
SEQ ID NO: 327
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0150-A04
SEQ ID NO: 320
SEQ ID NO: 93
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0150-A07
SEQ ID NO: 321
SEQ ID NO: 93
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0150-A12
SEQ ID NO: 320
SEQ ID NO: 93
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0150-D12
SEQ ID NO: 320
SEQ ID NO: 325
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0150-E01
SEQ ID NO: 320
SEQ ID NO: 93
SEQ ID NO: 335
SEQ ID NO: 341


807B-M0004-A03/M0150-E03
SEQ ID NO: 320
SEQ ID NO: 325
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0150-E04
SEQ ID NO: 320
SEQ ID NO: 327
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0150-E12
SEQ ID NO: 320
SEQ ID NO: 328
SEQ ID NO: 336
SEQ ID NO: 342


807B-M0004-A03/M0150-G01
SEQ ID NO: 320
SEQ ID NO: 329
SEQ ID NO: 337
SEQ ID NO: 341


807B-M0004-A03/M0151-A06
SEQ ID NO: 320
SEQ ID NO: 330
SEQ ID NO: 338
SEQ ID NO: 343


807B-M0004-A03/M0151-B12
SEQ ID NO: 320
SEQ ID NO: 325
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0151-C05
SEQ ID NO: 320
SEQ ID NO: 328
SEQ ID NO: 336
SEQ ID NO: 342


807B-M0004-A03/M0151-D09
SEQ ID NO: 322
SEQ ID NO: 325
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0151-F12
SEQ ID NO: 320
SEQ ID NO: 93
SEQ ID NO: 333
SEQ ID NO: 341


807B-M0004-A03/M0151-H05
SEQ ID NO: 320
SEQ ID NO: 331
SEQ ID NO: 339
SEQ ID NO: 341


807B-M0004-A03/M0153-D03
SEQ ID NO: 320
SEQ ID NO: 93
SEQ ID NO: 94
SEQ ID NO: 95


807B-M0004-A03/M0153-F07
SEQ ID NO: 320
SEQ ID NO: 327
SEQ ID NO: 333
SEQ ID NO: 341





Initial Name
HV-CDR1
HV-CDR2
LV-WholeAA
HV-WholeAA





807B-M0004-A03/M0148-E05
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 345
SEQ ID NO: 369


807B-M0004-A03/M0148-E08
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 346
SEQ ID NO: 369


807B-M0004-A03/M0149-D04
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 347
SEQ ID NO: 370


807B-M0004-A03/M0149-F02
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 348
SEQ ID NO: 370


807B-M0004-A03/M0149-G11
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 348
SEQ ID NO: 371


807B-M0004-A03/M0149-H07
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 350
SEQ ID NO: 370


807B-M0004-A03/M0149-H09
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 351
SEQ ID NO: 369


807B-M0004-A03/M0150-A04
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 352
SEQ ID NO: 369


807B-M0004-A03/M0150-A07
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 348
SEQ ID NO: 372


807B-M0004-A03/M0150-A12
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 354
SEQ ID NO: 369


807B-M0004-A03/M0150-D12
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 355
SEQ ID NO: 369


807B-M0004-A03/M0150-E01
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 356
SEQ ID NO: 369


807B-M0004-A03/M0150-E03
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 357
SEQ ID NO: 369


807B-M0004-A03/M0150-E04
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 358
SEQ ID NO: 369


807B-M0004-A03/M0150-E12
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 359
SEQ ID NO: 369


807B-M0004-A03/M0150-G01
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 360
SEQ ID NO: 369


807B-M0004-A03/M0151-A06
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 361
SEQ ID NO: 369


807B-M0004-A03/M0151-B12
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 362
SEQ ID NO: 369


807B-M0004-A03/M0151-C05
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 363
SEQ ID NO: 369


807B-M0004-A03/M0151-D09
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 362
SEQ ID NO: 370


807B-M0004-A03/M0151-F12
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 348
SEQ ID NO: 369


807B-M0004-A03/M0151-H05
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 366
SEQ ID NO: 369


807B-M0004-A03/M0153-D03
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 151
SEQ ID NO: 369


807B-M0004-A03/M0153-F07
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 368
SEQ ID NO: 369
















TABLE 40





Affinity matured clones of 807B-M0004-H03



















Initial Name
HV-CDR3
LV-CDR1
LV-CDR2
LV-CDR3





807B-M0004-H03/M0154-C07
SEQ ID NO: 373
SEQ ID NO: 388
SEQ ID NO: 381
SEQ ID NO: 377


807B-M0004-H03/M0154-D08
SEQ ID NO: 373
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0154-G05
SEQ ID NO: 373
SEQ ID NO: 390
SEQ ID NO: 383
SEQ ID NO: 379


807B-M0004-H03/M0154-G08
SEQ ID NO: 373
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0154-G11
SEQ ID NO: 374
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0154-H03
SEQ ID NO: 375
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0154-H06
SEQ ID NO: 373
SEQ ID NO: 391
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0155-C08
SEQ ID NO: 373
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0155-E10
SEQ ID NO: 376
SEQ ID NO: 392
SEQ ID NO: 384
SEQ ID NO: 380


807B-M0004-H03/M0155-F08
SEQ ID NO: 373
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0155-H06
SEQ ID NO: 373
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0155-H08
SEQ ID NO: 376
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0156-D01
SEQ ID NO: 373
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0156-G08
SEQ ID NO: 373
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0157-A08
SEQ ID NO: 373
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0157-D10
SEQ ID NO: 376
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0157-G08
SEQ ID NO: 373
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0157-G11
SEQ ID NO: 375
SEQ ID NO: 393
SEQ ID NO: 385
SEQ ID NO: 378


807B-M0004-H03/M0159-A09
SEQ ID NO: 373
SEQ ID NO: 394
SEQ ID NO: 386
SEQ ID NO: 378


807B-M0004-H03/M0159-H03
SEQ ID NO: 373
SEQ ID NO: 389
SEQ ID NO: 382
SEQ ID NO: 378


807B-M0004-H03/M0159-H07
SEQ ID NO: 373
SEQ ID NO: 395
SEQ ID NO: 387
SEQ ID NO: 380


807B-M0004-H03/M0157-F04
SEQ ID NO: 68
SEQ ID NO: 396
SEQ ID NO: 382
SEQ ID NO: 378





Initial Name
HV-CDR1
HV-CDR2
LV-WholeAA
HV-WholeAA





807B-M0004-H03/M0154-C07
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 400
SEQ ID NO: 397


807B-M0004-H03/M0154-D08
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 401
SEQ ID NO: 397


807B-M0004-H03/M0154-G05
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 402
SEQ ID NO: 397


807B-M0004-H03/M0154-G08
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 403
SEQ ID NO: 397


807B-M0004-H03/M0154-G11
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 404
SEQ ID NO: 217


807B-M0004-H03/M0154-H03
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 405
SEQ ID NO: 398


807B-M0004-H03/M0154-H06
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 406
SEQ ID NO: 397


807B-M0004-H03/M0155-C08
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 407
SEQ ID NO: 397


807B-M0004-H03/M0155-E10
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 408
SEQ ID NO: 399


807B-M0004-H03/M0155-F08
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 409
SEQ ID NO: 397


807B-M0004-H03/M0155-H06
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 410
SEQ ID NO: 397


807B-M0004-H03/M0155-H08
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 411
SEQ ID NO: 399


807B-M0004-H03/M0156-D01
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 412
SEQ ID NO: 397


807B-M0004-H03/M0156-G08
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 413
SEQ ID NO: 397


807B-M0004-H03/M0157-A08
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 414
SEQ ID NO: 397


807B-M0004-H03/M0157-D10
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 415
SEQ ID NO: 399


807B-M0004-H03/M0157-G08
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 416
SEQ ID NO: 397


807B-M0004-H03/M0157-G11
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 417
SEQ ID NO: 398


807B-M0004-H03/M0159-A09
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 418
SEQ ID NO: 397


807B-M0004-H03/M0159-H03
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 419
SEQ ID NO: 397


807B-M0004-H03/M0159-H07
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 420
SEQ ID NO: 397


807B-M0004-H03/M0157-F04
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 421
SEQ ID NO: 142
















TABLE 41





Affinity matured clones of 807B-M0009-F06



















Initial Name
HV-CDR3_1
LV-CDR1
LV-CDR2
LV-CDR3





807B-M0009-F06/M0173-F07
SEQ ID NO: 485
SEQ ID NO: 117
SEQ ID NO: 118
SEQ ID NO: 119


807B-M0009-F06/M0174-B01
SEQ ID NO: 486
SEQ ID NO: 117
SEQ ID NO: 118
SEQ ID NO: 119


807B-M0009-F06/M0174-B06
SEQ ID NO: 487
SEQ ID NO: 117
SEQ ID NO: 118
SEQ ID NO: 119


807B-M0009-F06/M0174-B08
SEQ ID NO: 488
SEQ ID NO: 117
SEQ ID NO: 118
SEQ ID NO: 119


807B-M0009-F06/M0175-A07
SEQ ID NO: 488
SEQ ID NO: 501
SEQ ID NO: 118
SEQ ID NO: 101


807B-M0009-F06/M0175-B01
SEQ ID NO: 485
SEQ ID NO: 502
SEQ ID NO: 497
SEQ ID NO: 101


807B-M0009-F06/M0175-B11
SEQ ID NO: 485
SEQ ID NO: 503
SEQ ID NO: 498
SEQ ID NO: 119


807B-M0009-F06/M0175-C07
SEQ ID NO: 488
SEQ ID NO: 501
SEQ ID NO: 118
SEQ ID NO: 101


807B-M0009-F06/M0175-D04
SEQ ID NO: 485
SEQ ID NO: 503
SEQ ID NO: 498
SEQ ID NO: 119


807B-M0009-F06/M0175-E04
SEQ ID NO: 485
SEQ ID NO: 503
SEQ ID NO: 118
SEQ ID NO: 492


807B-M0009-F06/M0175-E06
SEQ ID NO: 486
SEQ ID NO: 503
SEQ ID NO: 118
SEQ ID NO: 119


807B-M0009-F06/M0176-A06
SEQ ID NO: 485
SEQ ID NO: 504
SEQ ID NO: 118
SEQ ID NO: 101


807B-M0009-F06/M0176-C04
SEQ ID NO: 485
SEQ ID NO: 505
SEQ ID NO: 499
SEQ ID NO: 493


807B-M0009-F06/M0176-G02
SEQ ID NO: 485
SEQ ID NO: 225
SEQ ID NO: 118
SEQ ID NO: 101


807B-M0009-F06/M0177-E01
SEQ ID NO: 489
SEQ ID NO: 117
SEQ ID NO: 118
SEQ ID NO: 119


807B-M0009-F06/M0177-E05
SEQ ID NO: 486
SEQ ID NO: 117
SEQ ID NO: 118
SEQ ID NO: 119


807B-M0009-F06/M0177-E09
SEQ ID NO: 488
SEQ ID NO: 117
SEQ ID NO: 118
SEQ ID NO: 119


807B-M0009-F06/M0177-F09
SEQ ID NO: 488
SEQ ID NO: 503
SEQ ID NO: 118
SEQ ID NO: 494


807B-M0009-F06/M0177-H02
SEQ ID NO: 488
SEQ ID NO: 503
SEQ ID NO: 118
SEQ ID NO: 119


807B-M0009-F06/M0177-H05
SEQ ID NO: 489
SEQ ID NO: 117
SEQ ID NO: 118
SEQ ID NO: 119


807B-M0009-F06/M0177-H06
SEQ ID NO: 488
SEQ ID NO: 505
SEQ ID NO: 499
SEQ ID NO: 493


807B-M0009-F06/M0177-H07
SEQ ID NO: 488
SEQ ID NO: 506
SEQ ID NO: 118
SEQ ID NO: 495


807B-M0009-F06/M0178-A08
SEQ ID NO: 488
SEQ ID NO: 503
SEQ ID NO: 118
SEQ ID NO: 496


807B-M0009-F06/M0178-E02
SEQ ID NO: 486
SEQ ID NO: 507
SEQ ID NO: 118
SEQ ID NO: 494


807B-M0009-F06/M0178-H09
SEQ ID NO: 488
SEQ ID NO: 505
SEQ ID NO: 499
SEQ ID NO: 493





Initial Name
HV-CDR1
HV-CDR2
LV-WholeAA
HV-WholeAA





807B-M0009-F06/M0173-F07
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 508
SEQ ID NO: 500


807B-M0009-F06/M0174-B01
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 508
SEQ ID NO: 490


807B-M0009-F06/M0174-B06
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 508
SEQ ID NO: 491


807B-M0009-F06/M0174-B08
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 508
SEQ ID NO: 509


807B-M0009-F06/M0175-A07
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 227
SEQ ID NO: 509


807B-M0009-F06/M0175-B01
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 237
SEQ ID NO: 500


807B-M0009-F06/M0175-B11
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 242
SEQ ID NO: 500


807B-M0009-F06/M0175-C07
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 246
SEQ ID NO: 509


807B-M0009-F06/M0175-D04
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 264
SEQ ID NO: 500


807B-M0009-F06/M0175-E04
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 293
SEQ ID NO: 500


807B-M0009-F06/M0175-E06
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 301
SEQ ID NO: 490


807B-M0009-F06/M0176-A06
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 303
SEQ ID NO: 500


807B-M0009-F06/M0176-C04
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 313
SEQ ID NO: 500


807B-M0009-F06/M0176-G02
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 324
SEQ ID NO: 500


807B-M0009-F06/M0177-E01
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 332
SEQ ID NO: 510


807B-M0009-F06/M0177-E05
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 340
SEQ ID NO: 490


807B-M0009-F06/M0177-E09
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 344
SEQ ID NO: 509


807B-M0009-F06/M0177-F09
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 349
SEQ ID NO: 509


807B-M0009-F06/M0177-H02
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 353
SEQ ID NO: 509


807B-M0009-F06/M0177-H05
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 508
SEQ ID NO: 510


807B-M0009-F06/M0177-H06
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 364
SEQ ID NO: 509


807B-M0009-F06/M0177-H07
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 365
SEQ ID NO: 509


807B-M0009-F06/M0178-A08
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 367
SEQ ID NO: 509


807B-M0009-F06/M0178-E02
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 439
SEQ ID NO: 490


807B-M0009-F06/M0178-H09
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 442
SEQ ID NO: 509
















TABLE 42





Affinity matured clones of 807B-M0079-D10



















Initial Name
LV-CDR1
LV-CDR2
LV-CDR3
HV-CDR1





807B-M0079-D10/M0160-F02
SEQ ID NO: 422
SEQ ID NO: 434
SEQ ID NO: 444
SEQ ID NO: 78


807B-M0079-D10/M0160-F12
SEQ ID NO: 423
SEQ ID NO: 435
SEQ ID NO: 445
SEQ ID NO: 78


807B-M0079-D10/M0161-B04
SEQ ID NO: 424
SEQ ID NO: 434
SEQ ID NO: 446
SEQ ID NO: 78


807B-M0079-D10/M0161-G03
SEQ ID NO: 423
SEQ ID NO: 435
SEQ ID NO: 445
SEQ ID NO: 78


807B-M0079-D10/M0162-A11
SEQ ID NO: 425
SEQ ID NO: 435
SEQ ID NO: 447
SEQ ID NO: 78


807B-M0079-D10/M0162-D11
SEQ ID NO: 424
SEQ ID NO: 434
SEQ ID NO: 445
SEQ ID NO: 78


807B-M0079-D10/M0162-F04
SEQ ID NO: 426
SEQ ID NO: 436
SEQ ID NO: 448
SEQ ID NO: 78


807B-M0079-D10/M0164-B02
SEQ ID NO: 423
SEQ ID NO: 435
SEQ ID NO: 445
SEQ ID NO: 78


807B-M0079-D10/M0164-B03
SEQ ID NO: 425
SEQ ID NO: 435
SEQ ID NO: 447
SEQ ID NO: 78


807B-M0079-D10/M0164-C08
SEQ ID NO: 425
SEQ ID NO: 435
SEQ ID NO: 447
SEQ ID NO: 78


807B-M0079-D10/M0164-C09
SEQ ID NO: 427
SEQ ID NO: 437
SEQ ID NO: 449
SEQ ID NO: 78


807B-M0079-D10/M0164-C10
SEQ ID NO: 428
SEQ ID NO: 438
SEQ ID NO: 450
SEQ ID NO: 78


807B-M0079-D10/M0164-E05
SEQ ID NO: 424
SEQ ID NO: 434
SEQ ID NO: 446
SEQ ID NO: 78


807B-M0079-D10/M0165-A02
SEQ ID NO: 429
SEQ ID NO: 440
SEQ ID NO: 451
SEQ ID NO: 78


807B-M0079-D10/M0165-A06
SEQ ID NO: 430
SEQ ID NO: 441
SEQ ID NO: 452
SEQ ID NO: 78


807B-M0079-D10/M0165-B07
SEQ ID NO: 423
SEQ ID NO: 435
SEQ ID NO: 445
SEQ ID NO: 78


807B-M0079-D10/M0165-B08
SEQ ID NO: 428
SEQ ID NO: 438
SEQ ID NO: 450
SEQ ID NO: 78


807B-M0079-D10/M0165-D03
SEQ ID NO: 431
SEQ ID NO: 434
SEQ ID NO: 445
SEQ ID NO: 78


807B-M0079-D10/M0165-D05
SEQ ID NO: 428
SEQ ID NO: 438
SEQ ID NO: 450
SEQ ID NO: 78


807B-M0079-D10/M0165-D12
SEQ ID NO: 428
SEQ ID NO: 438
SEQ ID NO: 450
SEQ ID NO: 78


807B-M0079-D10/M0165-E01
SEQ ID NO: 426
SEQ ID NO: 436
SEQ ID NO: 448
SEQ ID NO: 78


807B-M0079-D10/M0166-A08
SEQ ID NO: 429
SEQ ID NO: 440
SEQ ID NO: 451
SEQ ID NO: 78


807B-M0079-D10/M0166-A09
SEQ ID NO: 424
SEQ ID NO: 434
SEQ ID NO: 446
SEQ ID NO: 78


807B-M0079-D10/M0166-B08
SEQ ID NO: 423
SEQ ID NO: 435
SEQ ID NO: 445
SEQ ID NO: 78


807B-M0079-D10/M0166-C08
SEQ ID NO: 430
SEQ ID NO: 441
SEQ ID NO: 452
SEQ ID NO: 78


807B-M0079-D10/M0166-D03
SEQ ID NO: 432
SEQ ID NO: 443
SEQ ID NO: 454
SEQ ID NO: 78


807B-M0079-D10/M0166-F03
SEQ ID NO: 423
SEQ ID NO: 435
SEQ ID NO: 445
SEQ ID NO: 78


807B-M0079-D10/M0166-F04
SEQ ID NO: 425
SEQ ID NO: 435
SEQ ID NO: 447
SEQ ID NO: 78


807B-M0079-D10/M0166-F07
SEQ ID NO: 433
SEQ ID NO: 434
SEQ ID NO: 445
SEQ ID NO: 78


807B-M0079-D10/M0166-F08
SEQ ID NO: 424
SEQ ID NO: 434
SEQ ID NO: 446
SEQ ID NO: 78





Initial Name
HV-CDR2
HV-CDR3
LV-WholeAA
HV-WholeAA





807B-M0079-D10/M0160-F02
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 455
SEQ ID NO: 146


807B-M0079-D10/M0160-F12
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 456
SEQ ID NO: 146


807B-M0079-D10/M0161-B04
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 457
SEQ ID NO: 146


807B-M0079-D10/M0161-G03
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 458
SEQ ID NO: 146


807B-M0079-D10/M0162-A11
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 459
SEQ ID NO: 146


807B-M0079-D10/M0162-D11
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 460
SEQ ID NO: 146


807B-M0079-D10/M0162-F04
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 461
SEQ ID NO: 146


807B-M0079-D10/M0164-B02
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 462
SEQ ID NO: 146


807B-M0079-D10/M0164-B03
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 463
SEQ ID NO: 146


807B-M0079-D10/M0164-C08
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 464
SEQ ID NO: 146


807B-M0079-D10/M0164-C09
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 465
SEQ ID NO: 146


807B-M0079-D10/M0164-C10
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 466
SEQ ID NO: 146


807B-M0079-D10/M0164-E05
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 467
SEQ ID NO: 146


807B-M0079-D10/M0165-A02
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 468
SEQ ID NO: 146


807B-M0079-D10/M0165-A06
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 469
SEQ ID NO: 146


807B-M0079-D10/M0165-B07
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 470
SEQ ID NO: 146


807B-M0079-D10/M0165-B08
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 471
SEQ ID NO: 146


807B-M0079-D10/M0165-D03
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 472
SEQ ID NO: 146


807B-M0079-D10/M0165-D05
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 473
SEQ ID NO: 146


807B-M0079-D10/M0165-D12
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 474
SEQ ID NO: 146


807B-M0079-D10/M0165-E01
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 475
SEQ ID NO: 146


807B-M0079-D10/M0166-A08
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 476
SEQ ID NO: 146


807B-M0079-D10/M0166-A09
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 477
SEQ ID NO: 146


807B-M0079-D10/M0166-B08
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 478
SEQ ID NO: 146


807B-M0079-D10/M0166-C08
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 479
SEQ ID NO: 146


807B-M0079-D10/M0166-D03
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 480
SEQ ID NO: 146


807B-M0079-D10/M0166-F03
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 481
SEQ ID NO: 146


807B-M0079-D10/M0166-F04
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 482
SEQ ID NO: 146


807B-M0079-D10/M0166-F07
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 483
SEQ ID NO: 146


807B-M0079-D10/M0166-F08
SEQ ID NO: 453
SEQ ID NO: 80
SEQ ID NO: 484
SEQ ID NO: 146
















TABLE 43





Light chain sequences of antibodies selected by VH-CDR3 spiking and light chain shuffling



















Initial Name
LV-FR1
LV-CDR1
LV-FR2
LV-CDR2





807B-M0004-A03/M0149-D04
QSVLTQSPSASGT
SGSNSNVGTKTVN
WYQVLPGTAPKLLIY
SNTQRPS



PGQRVTISC



807B-M0004-A03/M0149-F02
QSALTQPPSASGT
SGSSSNIGSNTVN
WYQQLPGTAPKLLIY
SNNQRPS



PGQRVTISC


807B-M0004-A03/M0150-A07
QSALTQPPSASGT
SGSSSNIGSNTVN
WYQQLPGTAPKLLIY
SNNQRPS



PGQRVTISC


807B-M0004-A03/M0150-E03
QYELTQPPSASGT
SGSSSNIGINTVN
WYQQLPGTAPKLLIY
SNNQRPS



PGQRVTISC


807B-M0004-A03/M0151-D09
QSALTQPPSASGT
SGSSSNIGINTVN
WYQQLPGTAPKLLIY
SNNQRPS



PGQRVTISC


807A-M0028-B02/M0169-F03
QDIQMTQSPSSLS
QASQDISNYLN
WYQQKPGKAPQRLIY
GASTVQS



ASVGDRVTITC


807A-M0028-B02/M0171-E03
QDIQMTQSPSSLS
RTSQDIGNHLA
WYQQKPGKAPQRLIR
EASILQS



ASVGDRVTITC


807A-M0028-B02/M0171-G02
QDIQMTQSPSSLS
RASQGITNYLA
WFQQKPGKAPKSLM
YGAYKLQY



ASVGDTVTITC


807A-M0028-B02/M0172-F07
QDIQMTQSPSSLS
RTSQGIRNHLG
WFQQKPGKAPQRLIR
EASILQS



ASVGDRVTITC


807B-M0004-H03/M0154-H06
QDIQMTQSPSSLS
RASRGVSTSLN
WYQIKPEKAPKLLIY
AASSLQS



ASVGDRVTITC


807B-M0004-H03/M0159-A09
QDIQMTQSPSSLS
RASQSIRSYLN
WFQQKPGKAPKLLIY
AASTLQS



ASVGDRVTITC


807A-M0028-B02/M0168-D10
QDIQMTQSPSSLS
RTSQDIRNHLG
WFQQKPGKAPQRLIR
EASILQS



ASVGDRVTITC


807B-M0004-A03/M0149-G11
QSALTQPPSASGT
SGSSSNIGSNTVN
WYQQLPGTAPKLLIY
SNNQRPS



PGQRVTISC















Initial Name
LV-FR3
LV-CDR3
LV-FR4







807B-M0004-A03/M0149-D04
GVPDRFSGSKSGTSASLAISG
AAWDDSLNGPV
FGGGTRVTVL




LQSEDEADYYC



807B-M0004-A03/M0149-F02
GVPDRFSGSKSGTSASLAISG
AAWDDSLNGPV
FGGGTKLTVL




LQSEDEADYYC



807B-M0004-A03/M0150-A07
GVPDRFSGSKSGTSASLAISG
AAWDDSLNGPV
FGGGTKLTVL




LQSEDEADYYC



807B-M0004-A03/M0150-E03
GVPDRFSGSKSGTSASLAISG
AAWDDSLNGPV
FGGGTKLTVL




LQSEDEADYYC



807B-M0004-A03/M0151-D09
GVPDRFSGSKSGTSASLAISG
AAWDDSLNGPV
FGGGTKLTVL




LQSEDEADYYC



807A-M0028-B02/M0169-F03
GVPSRFSGSGSGTEFTLTISS
QQYKTYPFT
FGQGTRLDIK




LQPDDFATYYC



807A-M0028-B02/M0171-E03
GVPSTFSGSGSGTEFTLTISS
QQYDAFPFT
FGQGTKLEIK




LQPEDFASYYC



807A-M0028-B02/M0171-G02
GVPTKFSGSGSGTDFTLTIRS
LQYQTYPFT
FGPGTKVDLK




LQPEDFATYYC



807A-M0028-B02/M0172-F07
GVPSTFSGSGSGTEFTLTISS
LQYDSFPYT
FGQGTKLEIK




LQPEDFATYYC



807B-M0004-H03/M0154-H06
GVPSRFSGSGSGTDFTLAITS
QQSYSTPRT
FGPGTKVEIK




LQPEDFATYYC



807B-M0004-H03/M0159-A09
GVPSRFSGSGSGTDFTLTISS
QQSYSTPRT
FGQGTKLEIK




LQPEDFATYYC



807A-M0028-B02/M0168-D10
GVPSTFYGSGYGREFTLTISS
LQYDSFPYT
FGQGTKLEIK




LQPEDFATYYC



807B-M0004-A03/M0149-G11
GVPDRFSGSKSGTSASLAISG
AAWDDSLNGPV
FGGGTKLTVL




LQSEDEADYYC

















TABLE 44





Heavy chain sequences of antibodies selected by VH-CDR3 spiking and light chain shuffling





















HV-




Initial Name
HV-FR1
CDR1
HV-FR2
HV-CDR2





807B-M0004-A03/M0149-D04
EVQLLESGGGLVQPGGSLRL
RYLMM
WVRQAPGKGLEWVS
VISPSGGRTWYADSVKG



SCAASGFTFS


807B-M0004-A03/M0149-F02
EVQLLESGGGLVQPGGSLRL
RYLMM
WVRQAPGKGLEWVS
VISPSGGRTWYADSVKG



SCAASGFTFS


807B-M0004-A03/M0150-A07
EVQLLESGGGLVQPGGSLRL
RYLMM
WVRQAPGKGLEWVS
VISPSGGRTWYADSVKG



SCAASGFTFS


807B-M0004-A03/M0150-E03
EVQLLESGGGLVQPGGSLRL
RYLMM
WVRQAPGKGLEWVS
VISPSGGRTWYADSVKG



SCAASGFTFS


807B-M0004-A03/M0151-D09
EVQLLESGGGLVQPGGSLRL
RYLMM
WVRQAPGKGLEWVS
VISPSGGRTWYADSVKG



SCAASGFTFS


807A-M0028-B02/M0169-F03
EVQLLESGGGLVQPGGSLRL
MYMMD
WVRQAPGKGLEWVS
SIWPSGGQTWYADSVKG



SCAASGFTFS


807A-M0028-B02/M0171-E03
EVQLLESGGGLVQPGGSLRL
MYMMD
WVRQAPGKGLEWVS
SIWPSGGQTWYADSVKG



SCAASGFTFS


807A-M0028-B02/M0171-G02
EVQLLESGGGLVQPGGSLRL
MYMMD
WVRQAPGKGLEWVS
SIWPSGGQTWYADSVKG



SCAASGFTFS


807A-M0028-B02/M0172-F07
EVQLLESGGGLVQPGGSLRL
MYMMD
WVRQAPGKGLEWVS
SIWPSGGQTWYADSVKG



SCAASGFTFS


807B-M0004-H03/M0154-H06
EVQLLESGGGLVQPGGSLRL
SYPMV
WVRQAPGKGLEWVS
GIWSSGGLTYYADSVKG



SCAASGFTFS


807B-M0004-H03/M0159-A09
EVQLLESGGGLVQPGGSLRL
SYPMV
WVRQAPGKGLEWVS
GIWSSGGLTYYADSVKG



SCAASGFTFS


807A-M0028-B02/M0168-D10
EVQLLESGGGLVQPGGSLRL
MYMMD
WVRQAPGKGLEWVS
SIWPSGGQTWYADSVKG



SCAASGFTFS


807B-M0004-A03/M0149-G11
EVQLLESGGGLVQPGGSLRL
RYLMM
WVRQAPGKGLEWVS
VISPSGGRTWYADSVKG



SCAASGFTFS















Initial Name
HV-FR3
HV-CDR3
HV-FR4







807B-M0004-A03/M0149-D04
RFTISRDNSKNTLYLQMNSLRAEDTA
SIASAGTDH
WGQGTLVTVSS




VYYCVR



807B-M0004-A03/M0149-F02
RFTISRDNSKNTLYLQMNSLRAEDTA
SIASAGTDH
WGQGTLVTVSS




VYYCVR



807B-M0004-A03/M0150-A07
RFTISRDNSKNTLYLQMNSLRAEDTA
SIAASRTDY
WGQGTLVTVSS




VYYCVR



807B-M0004-A03/M0150-E03
RFTISRDNSKNTLYLQMNSLRAEDTA
SIAADRTDY
WGQGTLVTVSS




VYYCVR



807B-M0004-A03/M0151-D09
RFTISRDNSKNTLYLQMNSLRAEDTA
SIASAGTDH
WGQGTLVTVSS




VYYCVR



807A-M0028-B02/M0169-F03
RFTISRDNSKNTLYLQMNSLRAEDTA
GVLLDK
WGQGTLVTVSS




VYYCAR



807A-M0028-B02/M0171-E03
RFTISRDNSKNTLYLQMNSLRAEDTA
GVLLDK
WGQGTLVTVSS




VYYCAR



807A-M0028-B02/M0171-G02
RFTISRDNSKNTLYLQMNSLRAEDTA
GILHDY
WGQGTLVTVSS




VYYCAR



807A-M0028-B02/M0172-F07
RFTISRDNSKNTLYLQMNSLRAEDTA
GVLFDN
WGQGTLVTVSS




VYYCAR



807B-M0004-H03/M0154-H06
RFTISRDNSKNTLYLQMNSLRAEDTA
ERSVAVFKARPRHYYYY
WGKGTTVTVSS




VYYCAR
MDV



807B-M0004-H03/M0159-A09
RFTISRDNSKNTLYLQMNSLRAEDTA
ERSVAVFKARPRHYYYY
WGKGTTVTVSS




VYYCAR
MDV



807A-M0028-B02/M0168-D10
RFTISRDNSKNTLYLQMNSLRAEDTA
GVLFDN
WGQGTLVTVSS




VYYCAR



807B-M0004-A03/M0149-G11
RFTISRDNSKNTLYLQMNSLRAEDTA
SIASARTDS
WGQGTLVTVSS




VYYCVR

## Claims

1. An isolated human, humanised or chimeric antibody or antibody fragment, which antibody or fragment:
(i) binds to a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 of the C-terminal domain of Apolipoprotein E (ApoE-CTD) or the amino acid sequence of a part thereof;
(ii) binds to human plaques; and
(iii) comprises:
(a) a heavy chain CDR3 region comprising the sequence shown in SEQ ID NO: 26, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, or SEQ ID NO: 512;
(b) a heavy chain CDR2 region comprising the sequence shown in SEQ ID NO: 25;
(c) a heavy chain CDR1 region comprising the sequence shown in SEQ ID NO: 24;
(d) a light chain CDR3 region comprising the sequence shown in SEQ ID NO: 35;
(e) a light chain CDR2 region comprising the sequence shown in SEQ ID NO: 34; and
(f) a light chain CDR1 region comprising the sequence shown in SEQ ID NO: 33.

2. An antibody or antibody fragment according to claim 1, wherein said heavy chain CDR3 region comprises an affinity matured variant of SEQ ID NO: 26 having the sequence shown in SEQ ID NO: 512.

3. An antibody or antibody fragment according to claim 1, wherein said heavy chain CDR3 region comprises an affinity matured variant of SEQ ID NO: 26 having the sequence shown in SEQ ID NO: 20.

4. An antibody or antibody fragment according to claim 1 wherein said heavy chain CDR3 region comprises the sequence shown in SEQ ID NO: 23.

5. An antibody or antibody fragment according to claim 2 wherein said heavy chain CDR3 region comprises an affinity matured variant of SEQ ID NO: 26 having the sequence shown in SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209 or SEQ ID NO: 210.

6. An antibody or antibody fragment according to claim 5, wherein said heavy chain CDR3 region comprises an affinity matured variant of SEQ ID NO: 26 having the sequence shown in SEQ ID NO: 207, SEQ ID NO: 208 or SEQ ID NO: 209.

7. An antibody or antibody fragment according to claim 1, wherein said polypeptide having the amino acid sequence of a part of SEQ ID NO: 1 comprises the sequence shown in SEQ ID NO: 5.

8. An antibody or antibody fragment according to claim 1, wherein said ApoE-CTD polypeptide is a recombinant polypeptide.

9. An antibody or antibody fragment according to claim 8 wherein said recombinant polypeptide is biotinylated.

10. An antibody or antibody fragment according to claim 1, which binds to said plaques in the presence of VLDL.

11. An antibody or antibody fragment according to claim 1, wherein said VLDL is present in human plasma.

12. An antibody or antibody fragment according to claim 11, which binds to the plaques in the presence of 25% plasma.

13. An antibody or antibody fragment according to claim 12, which binds to the plagues in the presence of from 25% to 50% plasma.

14. An antibody or antibody fragment according to claim 13, which binds to the plaques in the presence of 50% plasma.

15. An isolated antibody or antibody fragment which comprises:
(a) the heavy chain sequence shown in SEQ ID NO: 40 and the light chain sequence shown in SEQ ID NO: 518 and/or 519 or
(b) the heavy chain sequence shown in SEQ ID NO: 40 and the light chain sequence shown in SEQ ID NO: 520 and/or 521.

16. An isolated antibody or antibody fragment which comprises:
(a) the heavy chain CDR1 sequence shown in SEQ ID NO: 24, the heavy chain CDR2 sequence shown in SEQ ID NO: 25 and the heavy chain CDR3 sequence shown in any one of SEQ ID NOS: 207, 209 and 210; and
(b) the light chain ODR1, CDR2 and CDR3 sequences shown in SEQ ID NOS: 33, 34 and 35, SEQ ID NOS: 219, 247 and 269, SEQ ID NOS: 226, 252 and 275 or SEQ ID NOS: 218, 34 and 268.

17. An antibody or antibody fragment according to claim 16 wherein the heavy chain CDR3 region comprises the sequence shown in SEQ ID NO: 210 and the light chain comprises the sequences shown in SEQ ID NOS: 33, 34 and 35, the heavy chain CDR3 region comprises the sequence shown in SEQ ID NO: 209 and the light chain comprises the sequences shown in SEQ ID NOS: 219, 247 and 269 or SEQ ID NOS: 218, 34 and 268, or the heavy chain CDR3 region comprises the sequence shown in SEQ ID NO: 207 and the light chain comprises the sequence shown in SEQ ID NOS: 226, 252 and 275.

18. An antibody or antibody fragment according to claim 16, wherein the heavy chain comprises the sequence shown in any one of SEQ ID NO: 317, 318 or 319.

19. An antibody or antibody fragment according to claim 16, wherein the light chain comprises the sequence shown in SEQ ID NO: 43, 295, 294 or 304.

20. An antibody or antibody fragment according to claim 1, wherein said antibody is an lgG.

21. An antibody or antibody fragment according to claim 1, wherein said antibody fragment is a Fab fragment or scFv.

22. An antibody or antibody fragment according to claim 1, which is a monoclonal antibody.

23. An antibody or antibody fragment according to claim 1, which is a humanised antibody.

24. An antibody or antibody fragment according to claim 1, which is chimeric.

25. A pharmaceutical composition comprising an antibody or antibody fragment according to claim 1 and a pharmaceutically acceptable carrier or diluent.

26. A kit for detecting ApoE-CTD, which kit comprises an antibody or antibody fragment according to claim 1 and means for detecting said an antibody or antibody fragment.

27. An antibody or antibody fragment according to claim 1, which is a human antibody.