OA17139A - Antibodies to bradykinin B1 receptor ligands - Google Patents

Antibodies to bradykinin B1 receptor ligands Download PDF

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Publication number
OA17139A
OA17139A OA1201400447 OA17139A OA 17139 A OA17139 A OA 17139A OA 1201400447 OA1201400447 OA 1201400447 OA 17139 A OA17139 A OA 17139A
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OA
OAPI
Prior art keywords
seq
kallidin
antibody
des
antibodies
Prior art date
Application number
OA1201400447
Inventor
Han Li
Dorothea KOMINOS
Alla Pritsker
Matthew Davison
Nicolas Baurin
Govindan Subramanian
Xin Chen
Jie Zhang
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Sanofi
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Publication of OA17139A publication Critical patent/OA17139A/en

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Abstract

The disclosure provides antibodies that specifically bind to Kallidin or des-Arg 10-Kallidin. The disclosure also provides pharmaceutical compositions, as well as nucleic acids encoding anti-Kallidin or des- Arg10-Kallidin antibodies, recombinant expression vectors and host cells for making such antibodies, or fragments thereof. Methods of using antibodies of the disclosure to modulate Kallidin or des-Arg10- Kallidin activity or detect Kallidin or des-Arg10-Kallidin or, either in vitro or in vivo, are also provided by the disclosure. The disclosure further provides methods of making antibodies that specifically bind to des-Argg- Bradykinin and des-Arg10Kallidin-like peptide.

Description

ANTIBODIES TO BRADYKININ B1 RECEPTOR LIGANDS
RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/616,645, fiied March
28,2012, and French Patent Application Number 1350953, fiied February 4,2013. The contents of these applications are each hereby incorporated by reference in their entireties.
SEQUENCE LISTING
The Instant application contains a Sequence Listing which has been submitted In ASCII format via EFS-Web and Is hereby Incorporated by reference in its entirety. Said ASCII copy, created on March 15,2013, Is named 543895_SA9-O29PC_SeqJJst.txt and is 89,291 bytes in size.
BACKGROUND OF THE INVENTION
The bradykinin B1 receptor has been implicated in pathogenesis of Inflammatory disease and chronic pain. By modulating tissue Inflammation and rénal fibrosis, the B1 receptor has also been associated with pathogenesis of acute kidney Injury as well as chronic kidney diseases which are the main causes of end-stage rénal faiiure.
In humans, the major agonîsts ofthe bradykinin B1 receptor are the kinins. Kinîns are bioactive peptides produced from the proteolytic cleavage of kininogen proteins. The major kinin agonîsts of bradykinin B1 receptor are the decapeptide Kallidin, and the nonapeptide des-Argio-Kallidin (formed by the proteolytic cleavage the c-terminal arginine form Kallidin). Therefore, agents that can Inhibit the binding of Kallidin and des-Argio-Kallidin to the bradykinin B1 receptor hâve the potential to treat or prevent bradykinin B1 receptor-mediated pathologies.
Accordingly, there Is a need In the art for novel agents that Inhibit the binding of Kallidin and des-Argio-Kallidin to the bradykinin B1 receptor for use In the treatment of bradykinin B1 receptormediated human pathologies.
SUMMARY OF THE INVENTION
The présent invention provides antibodies, or antigen binding fragments thereof, that specifically bind Kallidin and des-Argio-Kallidin and prevent binding to the bradykinin B1 receptor. Such antibodies are particulariy useful for treating Kallidin and des-Argio-Kallidin-associated diseases or disorders (e.g., pain or fibrosis). The invention also provides pharmaceuticai compositions, as well as nucleic acids encoding anti-Kallidîn and des-Argio-Kallidin antibodies, recombinant expression vectors and host cells for making such antibodies, or fragments thereof. Methods of using antibodies, or fragments thereof, ofthe Invention to detect Kallidin and des-ArgioKallidin or to modulate Kallidin and des-Argio-Kallidin activity, either in vitro or in vivo, are also encompassed by the Invention. The Invention also provides methods of making antibodies that specifically bind to des-Args- Bradykinin and des-Argio-Kallidin-like peptide.
Accordingly, in one aspect the invention provides an isolated monoclonal antibody or antigen binding fragment thereof that:
a) specifically binds to Kallidin or des-Argio-Kallidin but not to Bradykinin or des-ArggBradyklnin;
b) specifically binds to Kallidin or des-Argio-Kallidin with a KD of less than 1x1010 M;
c) specifically binds to Kallidin or des-Argio-Kallidin with a Km of less than IxlO4 s*1; or
d) specifically binds to Kallidin or des-Argio-Kallidin and inhibits binding to the bradykinin B1 receptor.
In one embodiment, the antibody or antigen binding fragment thereof binds to the hl-terminal Lysine residue of Kallidin or des-Argio-Kallidin.
In another embodiment, the antibody or antigen binding fragment thereof inhibits the binding of Kallidin or des-Argio-Kallidin to a bradykinin-1 receptor.
In another embodiment the antibody or antigen binding fragment thereof binds specifically to mouse Kallidin-like peptide (KLP).
In another embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain variable domain comprising an HCDR3 amino acid sequence selected from the group consisting of:
a) SEQ ID NO: 7 [XiY X2 X3D XxHAM X5Y], wherein
Xi is Y, F or H,
Xds R, D, A, V, L, I, M, F, Y or W,
Xais Y, F, Wor H,
X< is D, E or Y, and,
Xî is D or E;
b) SEQ ID NO: 63 [XiEYDGX^fXaXiLDXsJ, wherein
XiisWor F,
X2 is N or no amino add;
XjisYorS,
X« is D or P, and
Xî is F or Y;
c) SEQID NO: 13;
d) SEQ ID NO: 32;
e) SEQID NO: 40;
f) SEQ ID NO: 47; and
g) SEQ ID NO: 55.
ι _
I
In another embodiment, the antibody or antigen binding fragment thereof comprises an HCDR2 amino add sequence selected from the group consisting of:
a) SEQ ID NO: 8 [YFX1PX2NGNTGYNQKFRG], wherein
Xi Is D, R, A, V, L, I, M, F, Y or W, and
X2isY, D, E, N, orQ;
b) SEQ ID NO: 64 [WX1DPENGDX2X3YAPKFQG], wherein
Xiisl, orV,
X2isT, or S, and
Xjis G, or D;
c) SEQ ID NO: 14
d) SEQ ID NO: 33;
e) SEQ IDNO: 41;
f) SEQ ID NO: 48; and
g) SEQ ID NO: 56.
In another embodiment, the antibody or antigen binding fragment thereof comprises an HCDR1 amino acid sequence selected from the group consisting of:
a) SEQ ID NO: 9 [GYSFTDYX1IY], wherein X1 is N, W or Y;
b) SEQ ID NO: 65 [ΰΡΝΙΚΟΥΥΧ,Η]. wherein X1 is L, or M;
c) SEQ IDNO: 15;
d) SEQ ID NQ: 34;
e) SEQ ID NO: 42;
f) SEQ ID NQ: 49; and
g) SEQ ID NQ: 57.
In another embodiment, the antibody or antigen binding fragment thereof comprises a light chain variable domain comprising an LCDR3 amino add sequence selected from the group consisting of:
a) SEQ ID NQ: 10 [QQ X1X2S X3P XÎTJ, wherein
X1 is Y, F or H,
XîlsY.F, HorW,
Xa Is Y, F, T or H, and,
XdsW, Y, F, HorL:
b) SEQ ID NQ: 66 [QX1X2X3SX4PX5T], wherein
X1 Is Q or N,
X2isY, F, DorH,
Xais Y, F, HorW,
Xds Y, F, T or H, and
Xs Is W, Y, F, H or L;
c) SEQ ID NO: 69 [XiQGTHFPYT], wherein X1 Is L or M;
d) SEQ ID NO: 16;
e) SEQ ID NO: 35;
f) SEQ ID NO: 43;
g) SEQ ID NO: 50; and
h) SEQ ID NO: 58.
In another embodiment, the antibody or antigen binding fragment thereof comprises an LCDR2 amino acid sequence selected from the group consisting of:
a) SEQ ID NO: 11 [WASTRXi], wherein Xi is E, D, Q or N;
b) SEQ ID NO: 67 [XiASTRX2], wherein
Xi is W or G, and
X2is E, D.Qor N;
c) SEQ ID NO: 17;
d) SEQ ID NO: 36;
e) SEQIDNO: 51;and
f) SEQ ID NO: 59.
In another embodiment, the antibody or antigen binding fragment thereof comprises an LCDR1 amino acid sequence selected from the group consisting of:
a) SEQ ID NO: 12 [KSSQSLL XiSSNQKN X2l_A], wherein
Xi isW, H, Y or F, and
X2 is H or Y;
b) SEQ ID NO: 68 [KSSQSLLX1X2SX3QX4NX5LA], wherein
Xi isW.H.Yor F,
X2 is S or G,
XaisNor D,
XdsKorR,
XsisHorY.
c) SEQ ID NO: 70 [KSSQSLLYSNGXiTYLN],wherein X, is K or E;
b) SEQ IDNO: 18;
c) SEQ ID NO: 37;
d) SEQ ID NO: 44;
e) SEQ ID NO: 52; and
f) SEQ ID NO: 60.
In another embodiment, the antibody or antigen binding fragment comprises a light chain variable domain comprising an LCDR3 amino acid sequence selected from the group consisting of:
a) SEQ ID NO: 10 [QQ Xi X2S XaP XxT], wherein
Xi is Y, F or H,
X2is Y, F, HorW,
Xais Y. F, T or H, and.
X4 is W. Y, F, H or L:
b) SEQ ID NO: 66 [QXiXzXjSXaPXsT], wherein
Xi is Q or N,
Xais Y, F, DorH,
XaisY, F, HorW,
XJs Y. F, T or H, and
XsisW. Y. F, HorL;
c) SEQ ID NO: 69 [XiQGTHFPYT], wherein Xi is L or M;
d) SEQID NO: 16;
e) SEQ ID NO: 35;
f) SEQ ID NO: 43;
g) SEQ ID NO: 50; and
h) SEQ ID NO: 58.
ln another embodiment, the antibody or antigen binding fragment thereof comprises an LCDR2 amino acid sequence selected from the group consisting of:
a) SEQ ID NO: 11 [WASTRXi], wherein Xi is E, D, Q or N;
b) SEQ ID NO: 67 [XiASTRXs], wherein
XiisWorG, and
XîisE, D.QorN;
c) SEQID NO: 17;
d) SEQ ID NO: 36;
e) SEQIDNO: 51;and
f) SEQ ID NO: 59.
ln another embodiment, the antibody or antigen binding fragment thereof comprises an LCDR1 amino acid sequence selected from the group consisting of:
a) SEQ ID NO: 12 [KSSQSLL XiSSNQKN XzLA], wherein
Xi isW.H, Y or F, and
XîisHorY;
b) SEQ ID NO: 68 [KSSQSLLX1X2SX3QX4NX5LA], wherein
Xi isW.H, Y or F,
XîisS orG,
X3 is N or D,
XdsKorR,
XsisHorY.
c) SEQ ID NO: 70 [KSSQSLLYSNGX1TYLN],wherein X1 is K or E;
b) SEQ ID NO: 18;
c) SEQ ID NO: 37;
d) SEQID NO: 44;
e) SEQ ID NO: 52; and
f) SEQ ID NO: 60.
ln another embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain variable région comprising the HCDR3, HCDR2 and HCDR1 région amino sequences set forth in SEQ ID NOs 13,14, and 15, respectively, and one or more amino acid substitutions at positions selected from the group consisting of H1, H5, H9, H11, H12, H16, H38, H40, H41, H43, H44, H66, H75, H79, H81, H82A, H83, H87, and H108 according to Kabat.
ln another embodiment, the antibody or antigen binding fragment thereof comprises a light chain variable région comprising the LCDR3, LCDR2 and LCDR1 région amino sequences set forth in SEQ ID NOs 16,17, and 18, respectively, and one or more amino add substitution at positions selected from the group consisting of L5, L9, L15, L18, L19, L21, L22, L43, L63, L78, L79, L83, L85, L100 and L104, according to Kabat ln another embodiment the antibody or antigen binding fragment thereof comprises a heavy chain variable région amino acid sequence with at least 90% identity to an amino add sequence selected from the group consisting of SEQ ID NOs: 19,20, 21,22, 24,25, 38,45, 53, and 61.
ln another embodiment the antibody or antigen binding fragment thereof comprises a light chain variable domain amino acid sequence with at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27,28, 29,29, 30, 31,39,46,54, and 62.
ln another embodiment, the antibody or antigen binding fragment thereof comprises a light chain variable région amino add sequence with at least 90% identity to an amino add sequence selected from the group consisting of SEQ ID NOs: 26,27, 28, 29, 29,30, 31,39,46,54, and 62.
ln another embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain variable domain comprising an amino add sequence selected from the group consisting of: SEQ ID NO: 19, 20. 21, 22, 24, 25, 38,45, 53, and 61.
ln another embodiment, the antibody or antigen binding fragment thereof comprises a light chain variable domain amino add sequence selected from the group consisting of: SEQ ID NO: 26, 27,
28, 29, 29, 30, 31, 39, 46, 54, and 62.
In another embodiment, the antibody or antigen binding fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 26,27,28,29,29,30,31,39,46,54, and 62.
In another embodiment, the antibody or antigen binding fragment thereof comprises the heavy chain and light chain variable région amino add sequences set forth In SEQ ID NO: 19 and 26, SEQ ID NO: 20 and 27, SEQ ID NO: 21 and 28; SEQ ID NO: 22 and 28; SEQ ID NO: 23 and 29; SEQ ID NO: 24 and 30; SEQ ID NO: 25 and 31; SEQ ID NO: 38 and 39, SEQ ID NO: 45 and 46, SEQ ID NO: 53 and 54, or SEQ ID NO: 61 and 62, respectively.
In another aspect, the invention provides an antibody, or antigen binding fragment thereof, that spedfically binds to Kallidin and des-Argio-Kallidin, wherein the antibody, or antigen binding fragment thereof, competes for binding to Kallidin and des-Argio-Kallidin with an antibody comprising the heavy chain and light chain variable région amino acid sequences set forth in SEQ 15 ID NO: 19 and 26, SEQ ID NO: 38 and 39, SEQ ID NO: 45 and 46, SEQ ID NO: 53 and 54, or SEQ
ID NO: 61 and 62, respectively.
In another aspect, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof that competes for binding to Kallidin or des-Argio-Kallidin with the antibody of any 20 one of the preceding daims, and does not bind to Bradykinin or desArgo-Bradykinin.
In another aspect, the invention provides an isolated monoclonal antibody or antigen binding fragment thereof that specificaliy binds to a conformational epitope of kallidin (KD) or desArglOKaliidîn (DAKD) which adopts a Pro4 kink conformation comprising a type II tight tum at Proline 4 of 25 the KD or DAKD). In one embodiment, the Pro 4 kink conformation of KD or DAKD further comprises amino acid repeats of a sigmoid shape which align the hydrophobie side chains of the amino acids In a spatially stacking mode. In another embodiment, the antibody or antigen binding fragment thereof comprises (a) specificaliy binds Kallidin or des-Argio-Kallidin but not to Bradykinin or des-Arg®- Bradykinin; b) spedfically binds to Kallidin or des-Argio-Kallidin with a KD 30 of less than 1x10*10 M; c) spedfically binds to Kallidin or des-Argio-Kallidin with a Koo of less than
1x104 s*1; or d) specificaliy binds to Kallidin or des-Argio-Kallidin and Inhibits binding to the bradykinin B1 receptor.
In another aspect, the antibody or antigen binding fragment ofthe invention is conjugated to a 35 diagnostic or therapeutic agent.
In another aspect, the invention provides isolated nucleic add encoding the amino add sequence ofthe antibody, or antigen binding fragment thereof, ofthe invention.
ln another aspect, the invention provides recombinant expression vector comprising the nucleic add of the Invention.
ln another aspect, the Invention provides a host cell comprising the recombinant expression vector 5 of the invention.
ln another aspect, the invention provides a method of produdng an antibody that binds specifically to Kallidin and des-Argio-Kallidin, comprising culturing the host cell of the invention under conditions such that an antibody that binds specifically to Kallidin and des-Argio-Kallidin is produced by the host cell.
ln another aspect, the invention provides a pharmaceutical composition comprising the antibody, or antigen binding fragment thereof, of the Invention and one or more pharmaceutically acceptable carriers.
ln another aspect, the invention provides a method for treating a disease or disorder Kallidin or des-Argio-Kallidin-associated disease or disorder, the method comprising administering to a subject in need of thereof the pharmaceutical composition of the invention.
ln one embodiment, the disease or disorder is chronic pain.
ln another aspect, the invention provides a method of gerterating an antibody that specifically binds to des-Argy Bradykinin and des-Argto-Kailidin-like peptide comprising: immunlzing an animal with an immunogen comprising a peptide, wherein the peptide consiste of the amino acid sequence set 25 forth in SEQ ID No.11, and wherein the amino terminal arginine of the peptide is indirectly coupled to a carrier moiety through a linker moiety, such that an antibody that specifically binds to des-ArggBradykinin, des-Argio-Kallidin and des-Argio-Kallidin-like peptide is produced by the Immune system of the animal.
ln another embodiment, the method further comprises îsotating from the animal, the antibody, a nucleic isolating encoding the antibody, or an immune cell expressing the antibody.
ln one embodiment, the carrier moiety is a protein. ln another embodiment, the protein is Keyhole limpet hemocyanin (KLH). ln another embodiment, wherein the linker moiety comprises [Gly-Gly35 Giy]n, wherein n is at least 1.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the results of ELISA assays demonstrating binding of EE1 antibody to kinin peptides.
Figure 2 depicts the results of differential scanning calorimetry measurements of antibody F151.
Figure 3 depicts amino acid sequence alignments of the variable régions of the murine and humanized F151 antibody. Ail identical residues are listed In the alignment, while homologous residues are identified by + sign and non-homologous residues are left blank. Figure disdoses SEQ ID NOS 19,24,26, and 30, respectively, in order of appearance.
Figure 4 depicts an électron density map of the antigen binding site of the
F151 antibody/kaliidin complex.
Figure 5 depicts an électron density map of the antigen binding site of the F151antibody/des-Arg10-kallidin complex.
Figure 6 depicts a ribbon and stick représentation of the Fv subunit of F151 bound to kaliidin.
Figure 7 depicts an amino acid sequence alignment of the light chain variable régions of exemplary murine anti-kailidin antibodies of the invention. Amino add residues that interact with kaliidin are marked with asterisks. Figure discloses SEQ ID NOS 134,125,124,123,126, and 131, respectively, in order of appearance.
Figure 8 depicts an amino acid sequence alignment of the heavy chain variable régions of exemplary murine anti-kailidin antibodies of the invention. Amino acid residues that interact with kaliidin are marked with asterisks. Figure disdoses SEQ ID NOS 135,120,119,118,121, and 122, respectively, in order of appearance.
Figure 9 depicts the results of in vivo experiments determining the effect of EE1 antibody on formalin-induced acute inflammatory pain.
Figure 10 depicts the results of in vivo experiments determining the effect of EE1 antibody on CFA-induced mechanical hypersensitivity.
Figure 11 depicts the results of in vivo experiments determining the effect of EE1 antibody on CFA-induced thermal hypersensitivity.
Figure 12 depicts the results of in vivo experiments determining the effect of EE1 antibody 30 on CCI-induced mechanical hypersensitivity.
Figure 13 depicts the results of in vivo experiments determining the effect of EE1 antibody on CCI-induced thermal hypersensitivity.
Figure 14 depicts schematic maps of VL and VH expression constructs for generating humanized F151 variant HC3a/LC3a with the restriction DNA endonudease sites presented as deduced sequences In bold and underlined. Panel A depicts the light chain and Panel B depicts the heavy chain. Figure disdoses SEQ ID NOS 30,24, and 136, respectively, In order of appearance.
Figure 15 depicts an alignment of the F151 heavy chain (A) and light chain (B) amino add sequences with the closest human germline amino acid sequences.
Figure 16 depicts an alignment of the F151 heavy chain (A) and light chain (B) with a heavy chain locus (1-08 & 1-18) and light chain (VOIV-B3) locus of the VH1 sub-family. CDR régions and Vernier régions are indicated ln boldface and humanizing mutations are underlined.
Figure 17 depicts (A) the secondary and (B) tertiary structure of the main chain polypeptide backbone conformation of kallidin (KD) as bound to F151 antibody which comprises a type II tight tum at Proline 4 (C). Figure discloses SEQ ID NOS 2,2, and 133, respectively, in order of appea rance.
DETAILED DESCRIPTION
The présent invention provides antibodies that specificaliy bind to Kallidin and des-ArgioKallidin and prevent binding to the bradykinin B1 receptor. Such antibodies are particularly useful for treating Kallidin and des-Argio-Kallidin -associated disease or disorders (e.g., pain). The invention also provides pharmaceutical compositions, as well as nucleic acids encoding antiKallidin and des-Argio-Kallidin antibodies, recombinant expression vectors and host cells for making such antibodies, or fragments thereof. Methods of using antibodies of the invention to detect Kallidin and des-Argio-Kallidin or to modulate Kallidin and des-Argio-Kallidin activity, either in vitro or in vfvo, are also encompassed by the invention.
I. Définitions ln order that the présent invention may be more readily understood, certain terms are first defined.
As used herein, the term Kallidin refers to a peptide comprising or consisting of the amino acid sequence KRPPGFSPFR (SEQ ID NO. 1).
As used herein, the term des-Argio-Kallidin refers to a peptide comprising or consisting of the amino acid sequence KRPPGFSPF (SEQ ID NO. 2).
As used herein, the term mouse Kallidin or Kallidin-like peptide refers to a peptide comprising or consisting of the amino add sequence RRPPGFSPFR (SEQ ID NO. 3)
As used herein, the term mouse des-Argio-Kallidin or des-ArgioKallidin-lîke peptide refers to a peptide comprising or consisting of the amino acid sequence RRPPGFSPF (SEQ ID NO. 4).
As used herein, the term Bradykinin refers to a peptide comprising or consisting of the amino add sequence RPPGFSPFR (SEQ ID NO. 5).
As used herein, the term des-Argo-Bradykinin refers to a peptide comprising or consisting of the amino add sequence RPPGFSPF (SEQ ID NO. 6).
As used herein, the term antibody refers to immunoglobulin molécules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable région (abbreviated Vh or VH)and a heavy chain constant région (Ch or CH). The heavy chain constant région comprises three domains, Ch1, Ch2 and Ch3. Each light chain comprises a light chain variable région (abbreviated Vl) and a light chain constant région (Cl or CL). The light chaln constant région comprises one domain (Cl1). The Vh and Vl régions can be further subdivided into régions of hypervariability, termed complementarity determining régions (CDRs), interspersed with régions that are more conserved, termed framework régions (FR). Each Vh and Vl is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
As used herein, the term antigen-binding fragment of an antibody includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifïcally binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molécules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Non-limiting examples of antigen-binding portions include: (i) Fab fragments; (ii) F(ab*)2 fragments; (üi) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molécules; (vï) dAb fragments; and (vii) minimal récognition units consisting ofthe amino acid residues that mimtc the hypervariable région of an antibody (e.g., an isolated complementarity determining région (CDR)). Other engineered molécules, such as diabodies, triabodies, tetrabodies and minibodies, are also encompassed within the expression antigen-binding fragment
As used herein, the term CDR or complementarity determining région means the noncontiguous antigen combining sites found within the variable région of both heavy and light chain polypeptides. These particular régions hâve been described by Kabat étal., J. Biol. Chem. 252,6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest (1991), and by Chothia et d., J. Mol. Biol. 196:901-917 (1987) and by MacCallum étal., J. Mol. Biol. 262:732-745 (1996) where the définitions include overiapplng or subsets of amino acid residues when compared against each other. The amino add residues which encompass the CDRs as defined by each of the above cited référencés are set forth for comparison. In an embodiment of the invention, the term CDR is a CDR as defined by Kabat, based on sequence comparisons.
As used herein the term framework (FR) amino add residues refers to those amino acids in the framework région of an lg chain, The term framework région or FR région as used herein, indudes the amino acid residues that are part of the variable région, but are not part ofthe CDRs (e.g., using the Kabat définition of CDRs). Therefore, a variable région framework is between about 100-120 amino adds in length but includes onlythose amino adds outside ofthe CDRs.
As used herein, the term specifïcally binds to refera to the ability of an antibody or an antigen-binding fragment thereof to bind to an antigen with an Kd of at least about 1 x 10~® M, 1 x
10-7 M, 1 x 10^ M, 1 x 10·’ M, 1 x 10-10 M, 1 x 10-11 M, 1 x 10-12 M, or more. The term also encompasses refera to the ability of an antibody or an antigen-binding fragment thereof to bind to an antigen with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen. It shall be understood, however, that an antibody, or an antigen-binding fragment thereof, Is capable of specifically binding to two or more antigens which are related in sequence (e.g., Kallidin or desArg1O-Kallidin and mouse Kallidin or des-Arg1 O-Kallidin).
As used herein, the term antigen refers to the binding site or epitope recognized by an antibody or antigen binding fragment thereof.
As used herein, the term vector is intended to refer to a nucleic acid molécule capable of transporting another nucleic add to which it has been linked. One type of vector is a plasmid, which refers to a drcular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous réplication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of réplication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon Introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as recombinant expression vectors (or simply, expression vectors). ln general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms, plasmid and vector may be used interchangeably. However, the invention Is intended to include such other forms of expression vectors, such as viral vectors (e.g., réplication détective retroviruses, adenoviruses and adeno-associated viruses), which serve équivalent functions.
As used herein, the term host cell Is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that this term Is intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding générations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still induded within the scope of the term host cell as used herein.
As used herein, the term treat, “treating, and “treatment refer to therapeutic or preventative measures described herein. The methods of treatment employ administration to a subject, an antibody or antigen binding fragment of the présent invention, for example, a subject having a Kallidin and des-Argio-Kallidin-associated disease or disorder (e.g. an inflammatory disease) or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected ln the absence of such treatment.
As used herein, the term Kallidin or des-Argio-Kallidin -associated disease or disorder includes disease states and/or symptoms associated with a disease state, where altered levels or activity of Kallidin or des-Argio-Kallidin are found. Exemplary Kallidin or des-Argio-Kallidinassociated diseases or disorders Include, but are not limited to, pain and fibrosîs.
As used herein, the term effective amount refers to that amount of an antibody or an antigen binding fragment thereof that binds Kallidin or des-Argio-Kallidin, which is sufficient to effect treatment, prognosis ordiagnosis of a Kallidin or des-Argio-Kallidin-associated disease or disorder, as described herein, when administered to a subject. A therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and âge ofthe subject, the severity ofthediseasecondition, the mannerofadministration andthe like, which can readily be determined by one of ordinary skill in the art. The dosages for administration can range from, for example, about 1 ng to about 10,000 mg, about 1 ug to about 5,000 mg, about 1 mg to about 1,000 mg, about 10 mg to about 100 mg, of an antibody or antigen binding fragment thereof, according to the invention. Dosage régiments may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (i.e., side effects) of an antibody or antigen binding fragment thereof are minimized or outweighed by the bénéficiai effects.
As used herein, the term subject Includes any human or non-human animal.
As used herein, the term epitope refera to an antigenic déterminant that interacts with a spécifie antigen binding site in the variable région of an antibody molécule known as a paratope. A single antigen may hâve more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may hâve different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatiallyjuxtaposed amino acids from different segments ofthe linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain.
It is noted here that, as used in this spécification and the appended claims, the singular forms a, an, and the include plural reference unless the context clearly dictâtes otherwise.
II. Anti-Kallldln or des*Argio-Kallldln Antibodies
In one aspect the Invention provides antibodies, or antigen binding fragments thereof, that specifically bind to Kallidin or des-Argio-Kallidin. Exemplary VH, VL and CDR amino acid sequences ofthe antibodies ofthe invention are setforth in Table 1.
Table 1. VH, VL and CDR amino acid sequences of exemplary anti-Kallidin or des-Argio-Kallidin antibodies.
Antibody Clone Sequence SEQ ID NO.
F151 HCDR3 consensus XiYXîXjDXîHAMXjY where: Xt is Y, F or H; X2 is R, D, A, V, L, I, M, F, Y or W,· X3 is Y, F, W or H; X< is D, E or Y; and Xs is D or E. 7
F151 HCDR2 consensus YFXiPX2NGNTGYNQKFRG where: Xi is D, R, A, V, L, I, M, F, Y or W; and 8
X2 is Y, D, E, N, or Q.
FI5I HCDRl consensus GYSFTDYXiIY Where Xi is N, W or Y. 9
FI5I LCDR3 consensus QQXiXîSXïPX^T where: Xi is Y, F or H; X2 is Y, F, H or W; Xs is Y, F, T or H; and X* is W, Y, F, H or L. to
Fl5l LCDR2 consensus WASTRXi where Xi is E, D, Q or N. 11
FI51 LCDRl consensus KSSQSLLXiSSNQKNX2LA where: Xi is W, H, Y or F; and X2 is H or Y. 12
FI5l HCDR3 YYRYDDHAMDY 13
Fl5l HCDR2 YFDPYNGNTGYNQKFRG 14
F15I HCDRl GYSFTDYNIY IS
FI5I LCDR3 QQYYSYPWT 16
FI5I LCDR2 WASTRES 17
FI5I LCDRl KSSQSLLYSSNQKNYLA 18
Fl5l VH ElQLQQSGPELVKPGTSVKVSCKASGYSFTDYNIYWVKQ SHGKSLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSST AFMH LSS LTS DDSAVYYCANYYRYD DHAMDYWGQGTSVT VSS I9
Fl5l Humanized HCl EIQLVQSGPEVKKPGASVKVSCKASGYSFTDYNIYWVKQ SPGKSLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSST AFMHLSSLTSEDSAVYYCANYYRYDDHAMDYWGQGTSVT VSS 20
FI5I Humanized HC2a QIQLVQSGAEVKKPGASVKVSCKASGYS FTDYNIYWVKQ SPGKGLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSST AYMHLSSLTSEESAVYYCANYYRYDDHAMDYWGQGTSVT VSS 21
FIS) Humanized HC2b QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVKQ SPGKGLEWIGYFDPYNGNTGYNEKFRGKATLTVDKSSST AYMHLSSLTSEESAVYYCANYYRYDDHAMDYWGQGTSVT VSS 22
F15I Humanized HC2c QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVKQ SPGKGLEWIGYFDPYNGNTGYNQKFRGKATLTVDKSSST AYMHLSSKTSEESAVYYCANYYRYDDHAMDYWGQGTSVT VSS 23
FI5I Humanized HC3a QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYNIYWVRQ APGQGLEWIGYFDPYNGNTGYNQKFRGRATLTVDKSTST AYMELRSLRSDDTAVYYCANYYRYDDHAMDYWGQGTLVT VSS 24
Fl5l Humanized HC3b QVQLVQSGAEVKKPGASVKVSCKASGYS FT DYNIYWVRQ APGQGLEWMGYFDPYNGNTGYNQKFRGRVTMTTDTSTST AYMELRSLRSDDTAVYYCANYYRYDDHAMDYWGQGTLVT VSS 25
FI5I VL DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYL AWYQQKPGQSPKPLIYWASTRESGVPDRFTGSGSGTDFT 26
LTISSVKAEDLAIYYCQQYYSYPWTFGGGTKLEIK
Fl5l Humanized LCl DIVMSQSPSSLAASVGDRVTMSCKSSQSLLYSSNQKNYL AWYQQKPGKSPKPLIYWASTRESGVPDRFTGSGSGTDFT LTISSVQAEDLA1YYCQQYYSYPWTFGGGTKLE1K 27
Fl5l Humanized LC2a DIVMTQSPSSLSASVGDRVTISCKSSQSLLYSSNQKNYL AWYQQKPGKSPKPLIYWASTRESGVPDRFSGSGSGTDFT LTISSVQAEDLATYYCQQYYSYPWTFGGGTKLEIK 28
Fl5l Humanized LC2b DIVMTQSPSSL5ASVGDRVTISCKSSQSLLYSSNQKNYL AWYQQKPGKSPKPLXYWASTRESGVPDRFSGSGSGTDFT LTISSVQAEDKATYYCQQYYSYPWTFGGGTKLEIK 29
Fl5l Humanized LC3a DIVMTQSPDSLAVSLGERATXNCKSSQSLLYSSNQKNYL AWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFT LTIS SLQAE DVAVYYCQQYYSYPWT FGQGTKVEIK 30
F151 Humanized LC3b DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYL AWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFT LT1SSLQAEDVAVYYCQQYYSYPWTFGQGTKVEIK 31
B2l HCDR3 WEYDGYYDLDY 32
B21 HCDR2 WIDPENGDTGYARKFQG 33
B2I HCDR1 GFNIKDYYLH 34
B21 LCDR3 LQGTHFPYT 35
B21 LCDR2 LVSKLDS 36
B21 LCDRI KSSQSLLYSNGKTYLN 37
B2I VH EVQLQQSGAELVRSGASVKLSCTASG ΓΝΙKDYYLHWVKQ RPEQGLEW1GWIDPENGDTGYARKFQGKATMTADTSSNT VYLHLSSLTSEDTAVYYFNAWEYDGYYDLDYWGQGTSVT VSS 38
B2I VL DWMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLN WLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTL ΚΙIRVEAEDLGVYYCLQGTH FPYT FGGGTKLEIK 39
C63 HCDR3 EDYGGDY 40
C63 HCDR2 EIRSKSNNYATHYAESVKG 41
C63 HCDR1 GFTFSNYWMN 42
C63 LCDR3 QQYYSYPYT 43
C63 LCDR2 WASTRES 17
C63 LCDRI KSSQSLLYSSDQRNYLA 44
C63 VH EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQ SPEKGLEWVAEIRSKSNNYATHYAESVKGRFTISRDDSK SSVYLQMNNLRAEDTGIYYCIGEDYGGDYWGQGTSVTVS S 45
C63 VL DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSDQRNYL AWYQQRSGQSPKLLIYWASTRESGVPDRFTGSGSGTDFT LTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIK 46
I22HCDR3 FEYDGNYSPLDF 47
122 HCDR2 WVDPENGDSDYAPKFQ 48
122 HCDR1 GFNIKDYYMH 49
122 LCDR3 QNDHSYPLT 50
122 LCDR2 GASTRES 51
I22LCDR1 KSSQSLLNSGNQKNYLA 52
I22VH EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQ RPEQGLEWIGWVDPENGDSDYAPKFQGKATMTADTSSNT VYLQFSSLTSEDTAVYYCNAFEYDGNYSPLDFWGQGTSV TVSS 53
I22 VL DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYL AWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFT LTIS SVQAEDLAVYYCQNDHSY PLT FGAGTKLELK 54
I54 HCDR3 FEYDGNYSPLDF 55
I54 HCDR2 WVDPENGDSDYAPKFQG 56
I54 HCDRl GFNIKDYYMH 57
154 LCDR3 MQGTHFPYT 58
154 LCDR2 LVSKLDS 59
I54 LCDR1 KSSQSLLYSNGETYLN 60
154 VH EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQ RPEQGLEWIGWVDPENGDSDYAPKFQGKATMTADTSSNT VYLQFSSLTSEDTAVYYCNAFEYDGNYSPLDFWGQGTSV TVSS 61
154 VL DWMTQTPLTLSVPIGQPASISCKSSQSLLYSNGETYLN WLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSRSGTDFTL KISRVESEDLGVYYCMQGTHFPYTFGGGTKLEIK 62
B21/122/154 HCDR3 consensus XiEYDGXïYXiXîLDXü where: Xi is W or F; X: is N or no amino acid; X3 is Y or S; Xî is D or P; and Xs is F or Y. 63
B21/122/I54 HCDR2 consensus WXiDPENGDXîXjYAPKFQG where: Xi is I, or V; X2 is T, or S; and Xi is G, or D. 64
B21/I22/154 HCDRl consensus GFNIKDYYXiH where Xi is L, or M. 65
F151/C63/122 LCDR3 consensus QXiXîXiSXiPXjT where: Xi is Q or N; X2 is Y, F, D or H; X3 is Y, F, H or W; X< is Y, F, T or H; and X5 is W, Y, F, H or L. 66
F151/C63/122 LCDR2 consensus XiASTRX2 where: Xi is W or G;,and 67
X2 is E, D, Q or N
FI5I/C63/I22 LCDR1 consensus KSSQSLLX1X2SX3QX4NXJLA where; Xi is W, H, Y or F; X2 is S or G; X3 is N or D; X« is K or R; Xs is H or Y. 68
B21/154 LCDR3 consensus X1QGTHFPYT where; Xi is L or M; 69
B21/154 LCDR2 both identical LVSKLDS 36
B2I/154LCDRI consensus KSSQSLLYSNGXiTYLN where: X! is K or E; 70
In certain embodiments, the antibody, or antigen binding fragment thereof, comprises one or more CDR région amino acid sequence selected from the group consisting of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 32, 33, 34, 35, 36, 37, 40, 41,42, 43, 44,47,48, 49, 50, 51 52, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67. 68, 69, and 70.
In other embodiments, the antibody, or antigen binding fragment thereof, comprises HCDR3, HCDR2 and HCDR1 région amino acid sequences selected from the group consisting of:
a) SEQ ID NO: 7,8, and 9;
b) SEQIDNO: 13,14,and 15;
c) SEQ ID NO: 32, 33, and 34;
d) SEQIDNO:40,41,and42;
e) SEQ ID NO: 47,48, and 49;
f) SEQ ID NO: 55,56, and 57; and
g) SEQ ID NO: 63,64, and 65, respectively.
In other embodiments, the antibody, or antigen binding fragment thereof, comprises the LCDR3, LCDR2 and LCDR1 région amino acid sequences selected from the group consisting of:
a) SEQIDNO: 10,11.and 12;
b) SEQIDNO: 16,17,and 18:
c) SEQ ID NO: 35, 36, and 37;
d) SEQIDNO:43,17.and44;
e) SEQIDNO:50,51.and52;
f) SEQ ID NO: 58,59, and 60;
g) SEQ ID NO: 66,67, and 68; and
h) SEQ ID NO: 69,25, and 70, respectively.
ln other embodiments, the antibody, or antigen binding fragment thereof, comprises the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1 région amino acid sequences selected from the group consisting of:
a) SEQ ID NO: 7,8,9,10,11, and 12;
b) SEQ ID NO: 13,14,15,16,17, and 18;
c) SEQ ID NO: 32, 33, 34, 35, 36 and 37;
d) SEQ ID NO: 40,41,42,43,17, and 44;
e) SEQ ID NO: 47,48,49, 50,51. and 52; and
f) SEQ ID NO: 55, 56,57,58, 59, and 60, respectively
In other embodiment, the Invention provides humanized antibodies, or antigen binding fragments thereof, comprising one or more CDR régions (or conservatively modified variants thereof) from the murine antibodies disclosed herein. Any method of humanization can be employed to generate the humanized antibodies of the Invention. Suitable methods are disclosed herein and specifically exemplified ln Example 4.
ln a one particular embodiment, the humanized antibody, or antigen binding fragment thereof comprises:
a heavy chain variable région comprising the HCDR3, HCDR2 and HCDR1 région amino sequences set forth in 13,14, and 15, respectively, and one or more amino add substitution at positions selected from the group consisting of H1, H5, H9, H11, H12, H16, H38, H40, H41, H43, H44, H66, H75, H79, H81, H82A, H83, H87, and H108; and/or a light chain variable région comprising the LCDR3, LCDR2 and LCDR1 région amino sequences setforth in 16,17, and 18, respectively, and one or more amino add substitution at positions selected from the group consisting of L5, L9, L15, L18, L19, L21, L22, L43, L63, L78, L79, L83, L85, L100 and L104 (according to the Kabat numbering convention).
ln other embodiments, the antibody, or antigen binding fragment thereof, comprises the VH région amino add sequences set forth in SEQ IDNO: 19,20, 21,22, 24,25,38,45,53, and/or 61.
In other embodiments, the antibody, or antigen binding fragment thereof, comprises the VL région amino acid sequences set forth in SEQ ID NO: 26, 27, 28, 29, 29, 30, 31, 39,46,54, and/or 62.
ln other embodiments, the antibody, or antigen binding fragment thereof, comprises the VH and VL région amino add sequences selected from the group consisting of: SEQ ID NO: 19 and 26, SEQ ID NO: 20 and 27, SEQ ID NO: 21 and 28; SEQ ID NO: 22 and 28; SEQ ID NO: 23 and 29; SEQ ID NO: 24 and 30; SEQ ID NO: 25 and 31; SEQ ID NO: 38 and 39, SEQ ID NO: 45 and 46, SEQ ID NO: 53 and 54, or SEQ ID NO: 61 and 62, respectively.
In certain embodiments, the antibody, or antigen binding fragment thereof, comprises one or more CDR région amino acid sequence selected from the group consisting of SEQ ID NO: 7, 8,
9, 10, 11, 12, 13, 14, 15,16,17,18, 32, 33, 34, 35, 36, 37,40, 41, 42, 43, 44, 47.48,49, 50, 51 52,
55, 56, 57, 58, 59 and 60, wherein the one or more CDR région amino acid sequences comprises at least one or more conservative amino add substitutions.
The présent Invention also encompasses conservative amino add substitutions in the CDR amino add sequences (e.g., SEQ ID NOs:, 8, 9.10,11,12,13,14,15,16,17,18,32,33,34, 35, 36, 37,40, 41,42, 43,44, 47, 48, 49, 50, 51 52, 55, 56, 57, 58, 59 and 60) of the antibodies of the invention, i.e., amino add sequence modifications which do not abrogate the binding of the antibody to the antigen, e.g., Kallidin or des-Arg1O-Kallidin. Conservative amino add substitutions include the substitution of an amino add in one dass by an amino add of the same class, where a class Is defined by common physicochemical amino add side chain properties and high substitution frequendes in homologous proteins found In nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. Six general dasses of amino acid side chains hâve been categorized and indude: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class lll (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class lll residue such as Asn, Gin, or Glu, Is a conservative substitution. Thus, a predicted nonessential amino add residue In an anti-Kallidin or des-Arg10-Kallidin antibody is preferably replaced with another amino acid residue from the same class. Methods of identifying amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art (see, e.g., Brummell étal., Biochem. 32:1180-1187 (1993); Kobayashi étal. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sd. USA 94:412-417(1997)).
ln another embodiment, the présent invention provides anti-Kallidin or des-Arg10-Kallidin antibodies, or antigen binding fragment thereof, that comprise a VH and/or VL région amino add sequence with about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identity to the VH région amino acid sequence set forth in SEQ ID NO: 19,20, 21, 22, 24, 25,38,45, 53, or 61, or the VL région amino add sequence set forth in SEQ ID NO: 26, 27,28,29,29,30,31,39,46,54, or 62, respectively.
ln another embodiment the présent invention provides anti-Kallidin or des-Arg10-Kallidin antibodies that bind to the same epitope and/or cross compete with an antibody, or antigen binding fragment thereof comprising the VH and VL région amino acid sequences set forth in SEQ ID NO: 19 and 25, SEQ ID NO: 38 and 39, SEQ ID NO: 45 and 46, SEQ ID NO: 53 and 54, or SEQ ID NO: 61 and 62, respectively. Such antibodies can be identified using routine compétition binding assays induding, for example, surface plasmon résonance (SPR)-based compétition assays.
ln certain embodiments, the antibodies of the invention bind a conformational epitope of kallidin (KD) or desArglO-Kallidin (DAKD) which adopts a Pro4 kink conformation. As depicted in
Figure 17, a hallmark of the Pro 4 kink conformation is a type II tight tum in the main chain polypeptide backbone of KD or DAKD at Proline 4. As known to those of skill in the art, a type II tight tum conformation comprises three residues (X1-X2-X3) with the carbonyl of residue
Xlformîng a hydrogen bond with the amide N of residue X3, which is typically a glydne (see
Richardson JS. The anatomy and taxonomy of protein structure. Adv Protein Chem. 1981 ;34:16717139
339, which is Incorporated by reference herein). Accordingly, in certain embodiments, a type ii tight tum conformation is formed by the Pro3-Pro4-Gly5 motif of KD or DADK. in more spécifie embodiments, the Pro 4 kink conformation is further defined by ali or substantially ail of the remaining amino acids of KD (1-2 and 6-9) or DAKD adopting repeats of a sigmoid shape which align the hydrophobie side chains in a spatially stacking mode.
III. Modified Antl-Kallldln or des^Argio-Kallidin antibodies in certain embodiments, anti-Kallidin or des-Arg10-Kallidin antibodies of the invention may comprise one or more modifications. Modified forms of anti-Kallidin or des-Arg10-Kallidin antibodies 10 of the invention can be made using any techniques known in the art.
I) Reducing Immunogenlclty
In certain embodiments, anti-Kallidin or des-Arg10-Kallidin antibodies, or antigen binding fragments thereof, of the invention are modified to reduce their immunogenîcity using art15 recognized techniques. For example, antibodies, or fragments thereof, can be chimericized, humanized, and/or deimmunized.
In one embodiment, an antibody, or antigen binding fragments thereof, of the invention may be chimeric. A chimeric antibody is an antibody in which different portions of the antibody are derived from different animal species, such as antibodies having a variable région derived from a 20 murine monoclonai antibody and a human Immunoglobulin constant région. Methods for produclng chimeric antibodies, or fragments thereof, are known in the art See, e.g., Monison, Science 229:1202 (1985); Oi étal., BioTechniques 4:214 (1986); Giîties étal., J. immunoi. Methods 125:191-202 (1989); U.S. Pat Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entireties. Techniques developed for the production of chimeric 25 antibodies (Monison ef a/., Proc. Natt Acad. Sd. 81:851-855 (1984); Neuberger ef al., Nature 312:604-608 (1984); Takeda étal., Nature 314:452-454 (1985)) may be employed forthe synthesis of said molécules. For example, a genetic sequence encoding a binding specificity of a mouse antiKallidin or des-Arg10-Kallidin antibody molécule may be fused together with a sequence from a human antibody molécule of appropriate biological activity. As used herein, a chimeric antibody is a 30 molécule in which different portions are derived from different animal species, such as those having a variable région derived from a murine monoclonai antibody and a human immunoglobulin constant région, e.g., humanized antibodies.
In another embodiment, an antibody, or antigen binding fragment thereof, of the invention ls humanized. Humanized antibodies, hâve a binding specificity comprising one or more complementarity determïning réglons (CDRs) from a non-human antibody and framework régions from a human antibody molécule. Often, framework residues In the human framework régions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify ι
framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et a/., U.S. Pat No. 5,585,089; Riechmann et et., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art induding, for exampie, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfadng (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. étal., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat No. 5,565,332).
In a particular embodiment, a humanization method is employed that is based on the impact of the molecular flexibility of the antibody during and at immune récognition (see W02009/032661, which is incorporated herein by reference in its entirety). Protein flexibility is related to the molecular motion ofthe protein molécule. Protein flexibility is the abilîty of a whole protein, a part of a protein or a single amino acid residue to adopt an ensemble of conformations which differ significantly from each other. Information about protein flexibility can be obtained by performing protein X-ray crystallography expérimente (see, for example, Kundu étal. 2002, Biophys J 83:723-732.), nuciear magnetic résonance expérimente (see, for example, Freedberg ef al., J Am Chem Soc 1998,120(31):7916-7923) or by running molecular dynamics (MD) simulations. An MD simulation of a protein is done on a computer and allows one to détermine the motion of ail protein atoms over a period of time by calculating the physical interactions of the atoms with each other. The output of a MD simulation Is the trajectory of the studied protein over the period of time ofthe simulation. The trajectory is an ensemble of protein conformations, also called snapshots, which are periodically sampled over the period ofthe simulation, e.g. every 1 picosecond (ps). It is by analyzing the ensemble of snapshots that one can quantify the flexibility of the protein amino acid residues. Thus, a flexible residue is one which adopte an ensemble of different conformations in the context of the polypeptide within which that residue résides. MD methods are known in the art, see, e.g., Brooks et al. Proteins: A Theoretical Perspective of Dynamics, Structure and Thermodynamics (Wiley, New York, 1988). Several software enable MD simulations, such as Amber (see Case étal. (2005) J Comp Chem 26:1668-1688), Charmm (see Brooks étal. (1983) J CompChem4:187-217; and MacKerell étal. (1998) inThe Encyclopédie of Computational Chemistry vol. 1:271-177, Schleyer ef al., eds. Chichesten John Wiley & Sons) or Impact (see Rizzo et al. J Am Chem Soc; 2000; 122(51):12898-12900.)
Most protein complexes share a relatively large and planar buried surface and it has been shown that flexibility of binding partners provides the origin for their plasticity, enabling them to conformationally adapt to each other (Structure (2000) 8, R137-R142). As such, examples of induced fit hâve been shown to play a dominant rote In protein-protein interfaces. In addition, there is a steadily increasing body of data showing that proteins actually bind ligands of diverse shapes sizes and composition (Protein Science (2002) 11:184-187) and that the conformational diversity appears to be an essential component ofthe abilîty to recognize different partners t « (Science (2003) 299,1362-1367). Flexible residues are involved in the binding of proteîn-protein partners (Structure (2006) 14,683-693).
The flexible residues can adopt a variety of conformations that provide an ensemble of interaction areas that are likely to be recognized by memory B cells and to trigger an immunogenic response. Thus, an antibody can be humanized by modifying a number of residues from the framework so that the ensemble of conformations and of récognition areas displayed by the modified antibody resemble as much as possible those adopted by a human antibody. That can be achieved by modifying a limited number of residues by: (1 ) building a homology model of the parent mAb and runnlng an MD simulation; (2) analyzing theflexible residues and identification ofthe most flexible residues of a non-human antibody molécule, as well as identilying residues or motifs likely to be a source of heterogeneity or of dégradation reaction; (3) identifying a human antibody which displays the most similar ensemble of récognition areas as the parent antibody; (4) determining the flexible residues to be mutated, residues or motifs likely to be a source of heterogeneity and dégradation are also mutated; and (5) checking for the presence of known T cell or B cell epitopes. The flexible residues can be found using an MD calculation as taught herein using an implicit solvent model, which accounts for the interaction of the water soivent with the protein atoms over the period of time of the simulation.
Once the set of flexible residues has been identified within the variable light and heavy chains, a set of human heavy and light chain variable région frameworks that closely resemble that of the antibody of interest are identified. That can be done, for example, using a BLAST search on the set of flexible residues against a database of antibody human germ line sequence. It can also be done by comparing the dynamics of the parent mAb with the dynamics of a library of germ line canonical structures. The CDR residues and neighboring residues are excluded from the search to ensure high affinity for the antigen Is preserved. Flexible residues then are replaced.
When several human residues show similar homologies, the sélection is driven also by the nature of the residues that are likely to affect the solution behavior of the humanized antibody. For instance, polar residues will be preferred in exposed flexible loops over hydrophobie residues. Residues which are a potential source of Instability and heterogeneity are also mutated even if there are found in the CDRs. That will include exposed methionines as sulfoxide formation can resuit from oxygen radicals, proteolytic cleavage of acid labile bonds such as those of the Asp-Pro di peptide (Drug Dev Res (2004) 61:137-154), deamidation sites found with an exposed asparagine residue followed by a small amino acid, such as Gly, Ser, Ala, H is, Asn or Cys (J Chromatog (2006) 837:35-43) and N-glycosylation sites, such as the Asn-X-Ser/Thr site. Typtcally, exposed methionines will be substituted by a Leu, exposed asparagines will be replaced by a glutamine or by an aspartate, or the subséquent residue will be changed. For the glycosylation site (Asn-XSer/Thr), either the Asn or the Ser/Thr residue will be changed.
The resulting composite antibody sequence is checked for the presence of known B cell or linear T-cell epitopes. A search is performed, for example, with the publicly available Immune Epitope Data Base (IEDB) (PLos Biol (2005) 3(3)e91). If a known epitope is found within the composite sequence, another set of human sequences Is retrieved and substituted. Thus, unlike the resurfacing method of U.S. Pat No. 5,639,641, both B-celî-mediated and T-cell-mediated immunogenic responses are addressed by the method. The method aiso avoids the Issue of ioss of activity that is sometimes observed with CDR grafting (U.S. Pat. No. 5,530,101). ln addition, stability and solubility issues also are consldered in the engineering and sélection process, resulting in an antibody that Is optlmized for low immunogenicity, high antigen affinrty and Improved biophysical properties.
ln some embodiments, de-immunization can be used to decrease the Immunogenicity of and antibody, or antigen binding fragment thereof. As used herein, the term de-immunization 10 includes alteration of an antibody, or antigen binding fragment thereof, to modify T cell epitopes (see, e.g., WO9852976A1, W00034317A2). For example, VH and VL sequences from the starting antibody may be analyzed and a human T cell epitope map may be generated from each V région showing the location of epitopes ln relation to complementarity-determining régions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are 15 analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody. A range of alternative VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated Into a range of Kallidin or des-Arg10-Kallidin-specific antibodies or fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for fonction. Typically, 20 between 12 and 24 variant antibodies are generated and tested. Complété heavy and light chain genes comprising modified V and human C régions are then cloned into expression vectors and the subséquent plasmids introduced Into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identifîed.
II) Effector Functions and Fc Modifications
Anti-Kallidin or des-Arg10-Kallidin antibodies of the invention may comprise an antibody constant région (e.g. an IgG constant région e.g., a human IgG constant région, e.g., a human IgG 1 or lgG4 constant région) which médiates one or more effector fonctions. For example, binding 30 of the C1 component of complément to an antibody constant région may activate the complément system. Activation of complément Is Important ln the opsonisation and lysis of cell pathogens. The activation of complément also stimulâtes the inflammatory response and may also be involved in autoimmune hypersensitivity. Further, antibodies bind to receptors on various cells via the Fc région, with a Fc receptor binding site on the antibody Fc région binding to a Fc receptor (FcR) on a 35 cell. There are a number of Fc receptors which are spécifie for different classes of antibody, Including IgG (gamma receptors), IgE (epsilon receptors), IgA (aipha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses Including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called
4 antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production. In preferred embodiments, the antibodies, or fragments thereof, ofthe invention bind to an Fc-gamma receptor. In alternative embodiments, anti-Kallidin or des-ArglO-Kallidïn antibodies ofthe invention may comprise a constant région which Is devoid of one or more effector functions (e.g., ADCC activity) and/or is unable to bind FcQ receptor.
Certain embodiments ofthe invention include anti-Kallidin or des-Arg10-Kallidin antibodies in which at least one amino add in one or more ofthe constant région domains has been deleted or otherwise altered so as to provide desired blochemlcal characteristics such as reduced or enhanced effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced sérum half-life, or increased sérum hatf-Iife when compared with a wtiole, unaltered antibody of approximately the same immunogenidty. For example, certain antibodies, or fragments thereof, for use in the diagnostic and treatment methods described herein are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain domains. For instance, in certain antibodies, one entire domain ofthe constant région ofthe modified antibody will be deleted, for example, ail or part ofthe CH2 domain will be deleted.
In certain other embodiments, anti-Kallidin or des-Arg10-Kallidin antibodies comprise constant régions derived from different antibody isotypes (e.g., constant régions from two or more ofa human lgG1, lgG2, lgG3, or lgG4). In otherembodiments, anti-Kallidin ordes-ArglO-Kallldin antibodies comprises a chimeric hinge (Le., a hinge comprising hinge portions derived from hinge domains of different antibody Isotypes, e.g., an upper hinge domain from an lgG4 molécule and an lgG1 middle hinge domain). In one embodiment an anti-Kallidin or des-Arg10-Kallidin antibodies comprises an Fc région or portion thereof from a human lgG4 molécule and a Ser228Pro mutation (EU numbering) inthe core hinge région ofthemolécule.
In certain anti-Kallidin or des-Arg10-Kallidin antibodies, the Fc portion may be mutated to Increase or decrease effector function using techniques known in the art For example, the délétion or Inactivation (through point mutations or other means) of a constant région domain may reduce Fc receptor binding ofthe circulating modified antibody thereby increasing tumor localization. In other cases it may be that constant région modifications consistent with the instant invention moderate complément binding and thus reduce the sérum half life and nonspecific association of a conjugated cytotoxin. Yet other modifications ofthe constant région may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or flexibility. The resulting physiological profile, bioavailability and other biochemlcal effects ofthemodifications, such astumorlocalization, biodistribution and sérum halflife, may easily be measured and quantified using well know immunological techniques without undue expérimentation.
In certain embodiments, an Fc domain employed in an antibody ofthe invention is an Fc variant As used herein, the term Fc variant refers to an Fc domain having at least one amino acid substitution relative to the wild-type Fc domain from which said Fc domain Is derived. For example, wherein the Fc domain Is derived from a human lgG1 antibody, the Fc variant of said human lgG1 Fc domain comprises at least one amino add substitution relative to said Fc domain.
The amino add substitution(s) of an Fc variant may be located at any position (i.e., any EU convention amino add position) within the Fc domain. In one embodiment, the Fc variant comprises a substitution at an amino add position located In a hinge domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino add position located in a CH2 domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino add position located in a CH3 domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino add position located in a CH4 domain or portion thereof.
The antibodies of the invention may employ any art-recognized Fc variant which is known to impart an improvement (e.g., réduction or enhancement) In effector function and/or FcR binding. Said Fc variants may indude, for example, any one of the amino add substitutions disdosed in 15 International PCT Publications W088/07089A1, WO96/14339A1, WO98/05787A1,
WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WOOO/32767A1, WO00/42072A2, WO02/44215A2, W002/060919A2, WO03/074569A2, W004/016750A2, W004/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2, WO04/099249A2, W005/040217A2, W005/070963A1, WOO5/077981A2, WO05/092925A2, WO05/123780A2,
WO06/019447A1, W006/047350A2, and WO06/085967A2 or U.S. Pat Nos. 5,648,260; 5,739,277;
5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784, each of which Is incorporated by reference herein. In one exemplary embodiment, an antibodyofthe invention maycomprise an Fc variant comprising an amino add substitution at EU position 268 (e.g., H268D or H268E). In another exemplary embodiment, an antibody of the invention may comprise an amino add substitution at EU position 239 (e.g., S239D or S239E) and/or EU position 332 (e.g., I332D or I332Q).
In certain embodiments, an antibody ofthe invention may comprise an Fc variant comprising an amino acid substitution which aiters the antigen-lndependent effector fonctions of the 30 antibody, In particular the circuiating half-life of the antibody. Such antibodies exhibit either increased or decreased binding to FcRn when compared to antibodies lacking these substitutions, therefore, hâve an increased or decreased half-life in sérum, respectively. Fc variants with improved affinity for FcRn are anticipated to hâve longer sérum half-lives, and such moiecules hâve usefol applications in methods oftreating mammalswhere long half-life ofthe administered 35 antibody is desired, e.g., to treat a chronic disease or disorder. In contrast, Fc variants with decreased FcRn binding affinity are expected to hâve shorter half-lives, and such moiecules are also usefol, for example, for administration to a mammal where a shortened circulation time may be advantageous, e.g. for In vivo diagnostic imaging or in situations where the starting antibody has toxic side effects when présent In the circulation for prolonged periods. Fc variants with decreased
FcRn binding affinity are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in prégnant women. In addition, other applications in which reduced FcRn binding affinity may be desired include those applications in which localization the brain, kidney, and/or liver is deslred. in one exemplary embodiment, the altered antibodies of the invention exhibit reduced transport across the epithelium of kidney glomerulî from the vasculature.
In another embodiment, the altered antibodies of the invention exhibit reduced transport across the biood brain barder (BBB) from the brain, into the vascular space. In one embodiment, an antibody with altered FcRn binding comprises an Fc domain having one or more amino acid substitutions within the FcRn binding loop of an Fc domain. The FcRn binding loop Is comprised of amino acid 10 residues 280-299 (according to EU numbering). Exemplary amino add substitutions which altered
FcRn binding activity are disdosed in International PCT Publication No. WO05/047327 which is incorporated by reference herein. in certain exemplary embodiments, the antibodies, or fragments thereof, of the invention comprise an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E and S304D (EU numbering).
In other embodiments, antibodies, for use In the diagnostic and treatment methods described herein hâve a constant région, e.g., an lgG1 or lgG4 heavy chain constant région, which is altered to reduce oreliminate glycosylation. For example, an antibody of the invention may also comprise an Fc variant comprising an amino add substitution which alters the glycosylation of the antibody. For example, said Fc variant may hâve reduced glycosylation (e.g., N- or O-linked glycosylation). In exemplary embodiments, the Fc variant comprises reduced glycosylation of the N-linked glycan normally found at amino add position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino add sequence NXT or NXS. In a particular embodiment, the antibody comprises an Fc variant with an amino acid substitution at amino add position 228 or 299 (EU numbering). In more particular embodiments, the antibody comprises an lgG1 or lgG4 constant région comprising an S228P and a T299A mutation (EU numbering).
Exemplary amino add substitutions which confer reduce or altered glycosylation are disclosed In International PCT Publication No. W005/018572, which is Incorporated by reference 30 herein. In preferred embodiments, the antibodies, or fragments thereof, of the invention are modified to eliminate glycosylation. Such antibodies, or fragments thereof, may be referred to as agly antibodies, or fragments thereof, (e.g. agly antibodies). While not being bound by theory, it is believed that agly antibodies, or fragments thereof, may hâve an improved safety and stability profile in vivo. Exemplary agly antibodies, or fragments thereof, comprise an aglycosylated Fc 35 région of an lgG4 antibody which is devold of Fc-effector function thereby elimînating the potentiel for Fc mediated toxicity to the normal vital organs that express Kaliidin or des-Arg10-Kallidin. In yet other embodiments, antibodies, or fragments thereof, of the invention comprise an altered glycan. For example, the antibody may hâve a reduced number of fucose residues on an N-glycan at Asn297 of the Fc région, i.e., is afucosylated. In another embodiment, the antibody may hâve an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc région.
III) Covalent Attachment
Anti-Kaltidin or des-Arg10-Kaliidin antibodies of the invention may be modified, e.g., by the covalent attachment of a molécule to the antibody such that covalent attachment does not prevent the antibody from specifically binding to its cognate epitope. For exampie, but not by way of limitation, the antibodies, or fragments thereof, of the invention may be modified by glycosylation, acétylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to spécifie chemical cleavage, acétylation, formylation, etc. Additionally, the dérivative may contain one or more non-classical amino acids.
Antibodies, or fragments thereof, of the invention may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (Including covalent and 15 non-covalent conjugations) to polypeptides or other compositions. For example, anti-Kallidin or des-Arg10-Kallidin antibodies may be recombinantly fused or conjugated to molécules useful as labels in détection assays and effector molécules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
Anti-Kallidin or des-Arg10-Kallidin antibodies may be fused to heterologous polypeptides to
Increase the In vivo half life or for use ln Immunoassays using methods known in the art. For example, in one embodiment, PEG can be conjugated to the anti-Kallidin or des-ArglO-Kallidin antibodies of the Invention to Increase their half-life in vivo. Leong, S. R., étal.. Cytokine 16:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002); or Weiref al, Biochem. Soc. Transactions 30:512 25 (2002).
Moreover, anti-Kallidin or des-Arg10-Kallidin antibodies of the Invention can be fused to marker sequences, such as a peptide to facilitate their purification or détection, ln preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide (SEQ ID NO: 137), such as the tag provided ln a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), 30 among others, many of which are commercially availabie. As described in Gentz et al, Proc. Natl.
Acad. Sel. USA 86:821-824 (1989), for Instance, hexa-histidine (SEQ ID NO: 137) provides for convenient purification of the fusion protein. Other peptide tags useful for purification Include, but are not limited to, the HA tag, which corresponds to an epitope derived from the Influenza hemagglutinin protein (Wilson étal, Cell 37:767 (1984)) and the flag tag.
Anti-Kallidin or des-Arg10-Kallidin antibodies of the invention may be used in nonconjugated form or may be conjugated to at least one of a variety of molécules, e.g., to improve the therapeutic properties of the molécule, to facilitate target détection, or for imaging or therapy of the patient. Anti-Kallidin or des-Arg10-Kallidin antibodies of the invention can be labeled or conjugated either before or after purification, when purification is performed. ln particular, antï- Kallidin or des17139
Arg10-Kallidin antibodies of the invention may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
The présent Invention further encompasses anti-Kallidin or des-Arg10-Kallidin antibodies of the invention conjugated to a diagnostic or therapeutic agent The anti-Kallidin or des-Arg10Kallidin antibodies can be used diagnostically to, for example, monltor the development or progression of a immune cell disorder (e.g., CLL) as part of a clinical testing procedure to, e.g., détermine the efficacy of a given treatment and/or prévention regimen. Détection can be facilitated by coupling the anti-Kallidin or des-Arg10-Kallidin antibodies to a détectable substance. Examples 10 of détectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron émission tomographies, and nonradioactive paramagnetic métal ions. See, for example, U.S. Pat No. 4,741,900 for métal ions which can be conjugated to antibodies for use as diagnostics according to the présent invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamlne fluorescein, dansyi chloride or phycoerythrin; an example of a luminescent material Includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 1251,1311, 111lnor99Tc.
Anti-Kallidin ordes-Arg10-Kallidin antibodies for use in the diagnostic and treatment methods disclosed herein may be conjugated to cytotoxins (such as radioisotopes, cytotoxic drugs, or toxins) therapeutic agents, cytostatic agents, biological toxins, prodrugs, peptides, proteins, 25 enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, immunologically active ligands (e.g., lymphokines or other antibodies wherein the resulting molécule binds to both the neoplastic cell and an effector cell such as a T cell), or PEG.
ln another embodiment, an anti-Kallidin or des-Arg10-Kallidin antibody for use în the diagnostic and treatment methods disclosed herein can be conjugated to a molécule that 30 decreases tumor cell growth. ln other embodiments, the disclosed compositions may comprise antibodies, or fragments thereof, coupied to drugs or prodrugs. Still other embodiments of the présent invention comprise the use of antibodies, or fragments thereof, conjugated to spécifie biotoxins or their cytotoxic fragments such as ricin, gelonin. Pseudomonas exotoxin or diphtheria toxin. The sélection of which conjugated or unconjugated antibodyto use wili dépend on the type 35 and stage of cancer, use of adjunct treatment (e.g., chemotherapy or extemal radiation) and patient condition. It wili be appreciated that one skilled In the art could readily make such a sélection in view of the teachings herein.
It wili be appreciated that, in previous s tu d tes, anti-tumor antibodies labeled with isotopes hâve been used successfully to destroy tumor cells ln animal models, and in some cases in humans. Exemplary radioisotopes include: 90Y, 1251,1311,1231,1111n, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re. The radionuclides act by producing ionizing radiation which causes multiple strand breaks In nuclear DNA, leading to cell death. The Isotopes used to produce therapeutic conjugates typically produce high energy alpha- or beta-particles which hâve a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They hâve little or no effect on non-localized cells. Radionuclides are essentially non-immunogenic.
IV. Expression of Antl-Kallldln or des-Arg10-Kallldln Antibodies, or Antigen Binding Fragments Thereof
Following manipulation ofthe isolated geneticmaterial to provide anti-Kallidin ordes-Arg10Kallidin antibodies ofthe Invention as set forth above, the genes are typically Inserted In an expression vector for introduction into host cells that may be used to produce the desired quantity ofthe claimed antibodies, or fragments thereof.
The term vector or expression vector is used herein for the purposes of the spécification and claims, to mean vectors used in accordance with the présent invention as a vehicle for Introducing into and expressing a desired gene in a cell. As known to those skilled in the art, such vectors may easily be selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the Instant invention will comprise a sélection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
Numerous expression vector Systems may be employed for the purposes of this invention. For example, one class of vector utilizes DNA éléments which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others involve the use of polycistronic Systems with internai ribosome binding sites. Additionally, cells which hâve integrated the DNA Info their chromosomes may be selected by introducing one or more markers which allow sélection of transfected host cells. The marker may provide for prototrophy to an auxotrophîc host, biocide résistance (e.g., antibiotics) or résistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional éléments may also be needed for optimal synthesis of mRNA. These éléments may include signal sequences, splice signais, as well as transcriptional promoters, enhancers, and termination signais. In particulariy preferred embodiments the cloned variable région genes are inserted into an expression vector along with the heavy and light chain constant région genes (preferably human) synthetic as discussed above.
In other preferred embodiments the anti-Kallidin or des-Arg10-Kallidin antibodies, or fragmentsthereof, ofthe invention may be expressed using polycistronicconstruis. In such expression Systems, multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct These Systems advantageously use an
Internai ribosome entry site (IRES) to provide relatively high levels of polypeptides of the Invention In eukaryotic host cells. Compatible IRES sequences are disclosed In U.S. Pat No. 6,193,980, which is incorporated by reference herein. Those skilled in the art will appreciate that such expression Systems may be used to effectively produce the full range of polypeptides disclosed in the instant application.
More generally, once a vector or DNA sequence encoding an antibody, or fragment thereof, has been prepared, the expression vector may be introduced into an appropriate host cell. That is, the host cells may be transformed. Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate précipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. Mammalian Expression Vectors Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Most preferably, plasmid introduction into the host is via electroporation. The transformed cells are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis. Exemplary assay techniques include enzyme-linked Immunosorbent assay (ELISA), radioimmunoassay (RIA), orflourescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
As used herein, the term transformation shall be used in a broad sense to refer to the introduction of DNA into a récipient host cell that changes the génotype and consequentiy results In a change In the récipient cell.
Along those same lines, host cells refers to cells that hâve been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of processes for Isolation of polypeptides from recombinant hosts, the terms cell and cell culture are used interchangeably to dénoté the source of antibody unless it is clearty specifîed otherwise. In other words, recovery of polypeptide from the cells may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
In one embodiment, the host cell line used for antibody expression Is of mammalian origin; those skilled In the art can détermine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a dérivative of CVI with SV40T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothélial cells), RAJI (human lymphocyte), 293 (human kidney). In one embodiment, the cell line provides for altered glycosylation, e.g., afacosylation, of the antibodyexpressed therefrom (e.g., PER.C6.RTM. (Crucell) or FUT8-knock-out CHO cell lines (Potelligent.RTM. Cells) (Biowa, Princeton, N.J.)). In one embodiment NS0 cells may be used. CHO cells are particulariy preferred. Host cell lines are typlcally available from commercial services, the American Tissue Culture Collection or from published literature.
ln vitro production allows scale-up to give large amounts ofthe desired polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions are known In the art and Include homogeneous suspension culture, e.g. in an airiift reactor or ln a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fi bers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, lon-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-)affinity chromatography.
Genes encoding the anti-Kallidin or des-Arg10-Kallidin antibodies, or fragments thereof, of the invention can also be expressed non-mammalian cells such as bacterla or yeast or plant cells. ln this regard it wili be appredated that various unicellular non-mammalian microorganisms such as bacteria can also be transformed; i.e. those capable of being grown in cultures or fermentation. Bacteria, which are susceptible to transformation, include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It wili further be appredated that, when expressed in bacteria, the polypeptides can become part of indusion bodtes. The polypeptides must be isolated. purified and then assembled into functional molécules.
ln addition to prokaryotes, eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb étal.. Nature, 282:39 (1979); Kingsman étal., Gene, 7:141 (1979); Tschemper étal., Gene, 10:157 (1980)) is commonly used. This plasmid already contains the TRP1 gene which provides a sélection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence ofthe trpl lésion as a characteristic ofthe yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
V. Pharmaceutical Formulations and Methods of Administration of Anti-Kallidin or desArg10-Katlldln Antibodies.
in another aspect, the invention provides pharmaceutical compositions comprising an antiKallidin or des-Arg10-Kallidin antibody, or fragment thereof.
Methods of preparing and administering antibodies, or fragments thereof, ofthe invention to a subject are well known to or are readily determined by those skilled in the art. The route of administration ofthe antibodies, or fragments thereof, ofthe invention may be oral, parentéral, by inhalation or topical. The term parentéral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The intravenous, intraarterial, subcutaneous and intramuscular forms of parentéral administration are generally preferred. While ali these forms of administration are cleariy contemplated as being within the scope of the invention, a form for administration would be a solution for injection, in particular for
Intravenous or intraarterial injection or drip. Usually, a suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. However, in other methods compatible with the teachlngs herein, the polypeptides can be delivered directly to the site of the adverse cellular population thereby Increasing the exposure of the diseased tissue to the therapeutic agent
Préparations for parentéral administration include stérile aqueous or non-aqueous solutions, suspensions, and émulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, émulsions or suspensions, including saline and buffered media, ln the subject invention, pharmaceutically acceptable carriers Indude, but are not limited to, 0.01-0.1 M and preferably 0.05M phosphate buffer or 0.8% saline. Other common parentéral vehicles include sodium phosphate solutions, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehides indude fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be présent such as for example, antimlcrobials, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions suitable for injectable use indude stérile aqueous solutions (where water soluble) or dispersions and stérile powders for the extemporaneous préparation of stérile Injectable solutions or dispersions, ln such cases, the composition must be stérile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, éthanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fîuidity 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. Prévention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phénol, ascorblc acid, thimerosal and the like. ln many cases, it will be préférable to Include isotonie agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
ln any case, stérile Injectable solutions can be prepared by Incorporating an active compound (e.g., an antibody by itself or in combination with other active agents) ln the required amount in an appropriate solvent with one or a combination of ingrédients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a stérile vehicle, which contains a basic dispersion medium and the required other ingrédients from those enumerated above. ln the case of stérile powders for the préparation of stérile injectable solutions, the preferred methods of préparation are vacuum drying and freeze17139 drying, which yields a powder of an active ingrédient plus any additional desired ingrédient from a previously sterile-filtered solution thereof. The préparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptie conditions according to methods known ln the art Further, the préparations may be packaged and sold ln the form of a kit such as those described in co-pending U.S. Ser. No. 09/259,337 and U.S. Ser. No. 09/259,338 each of which is incorporated herein by reference. Such articles of manufacture will preferably hâve labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to autoimmune or neoplastic disorders.
Effective doses ofthe stabilized antibodies, or fragments thereof, ofthe présent invention, for the treatment of the above described conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other médications adminlstered, and whether treatment is prophyiactic or therapeutic. Usually, the patient is a human, but non-human mammals including transgenic mammals can also be treated. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
For passive immunization with an antibody of the Invention, the dosage may range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), ofthe host body weight For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg. Doses Intermediate in the above ranges are also intended to be within the scope of the invention,
Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment entalls administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment régimes entaïl administration once per every two weeks or once a month or once every 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on altemate days or 60 mg/kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered may fail within the ranges indicated.
Antibodies, or fragments thereof, ofthe invention can be adminlstered on multiple occasions. Intervals between single dosages can be, e.g., daily, weekly, monthly or yeariy. Interval s can also be Irregular as indicated by measuring blood levels of polypeptide or target molecuie in the patient, ln some methods, dosage is adjusted to achieve a certain plasma antibody or toxin concentration, e.g., 1-1000 ug/ml or 25-300 ug/ml. Altematively, antibodies, or fragments thereof, can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient In general, humanized antibodies show the longest half-life, followed by chimeric antibodies and nonhuman antibodies. In one embodiment, the antibodies, or fragments thereof, of the invention can be administered in unconjugated form. ln another embodiment, the antibodies of the Invention can be administered multiple times In conjugated form. In still another embodiment, the antibodies, or fragments thereof, of the Invention can be administered in unconjugated form, then in conjugated form, or vise versa.
The dosage and frequency of administration can vary depending on whether the treatment
Is prophylactic or therapeutic. In prophylactic applications, compositions containing the présent antibodies or a cocktail thereof are administered to a patient not already In the disease state to enhance the patients résistance. Such an amount Is defined to be a prophylactic effective dose. In this use, the précisé amounts agaln dépend upon the patients state of health and general Immunity, but generally range from 0.1 to 25 mg per dose, especially 0.5 to 2.5 mg per dose. A relatively low dosage is administered at relatively infrequent intervals over a long period of time.
Some patients continue to receive treatment for the rest of their lives.
In therapeutic applications, a relatively high dosage (e.g., from about 1 to 400 mg/kg of antibody per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxin-drug conjugated molécules) at relatively 15 short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complété amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic régime.
In one embodiment, a subject can be treated with a nucleic add molecuie encoding a polypeptide ofthe invention (e.g., in a vector). Doses fornudeic acids encoding polypeptides range 20 from about 10 ng to 1 g, 100 ng to 100 mg, 1 ug to 10 mg, or 30-300 ug DNA per patient. Doses for infectious viral vectors vary from 10-100, or more, virions per dose.
Therapeutic agents can be administered by parentéral, toplcal, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment intramuscular injection or intravenous infusion are 25 preferred for administration of a antibody of the invention. In some methods, therapeutic antibodies, or fragments thereof, are injected directly into the cranium. In some methods, antibodies, or fragments thereof, are administered as a sustained release composition or device, such as a Medipad™ device.
Agents ofthe invention can optionally be administered In combinationwith other agents that 30 are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic). Preferred additional agents are those which are art recognized and are standardly administered for a particular disorder.
Effective single treatment dosages (i.e., therapeutically effective amounts) of 90Y-labeled antibodies of the invention range from between about 5 and about 75 mCi, more preferably 35 between about 10 and about 40 mCi. Effective single treatment non-marrow ablative dosages of 1311-labeled antibodies range from between about 5 and about 70 mCi, more preferably between about 5 and about 40 mCi. Effective single treatment ablative dosages (i.e., may require autologous bone marrow transplantation) of 131 l-labeled antibodies range from between about 30 and about 600 mCi, more preferably between about 50 and less than about 500 mCi. In conjunction with a chimeric modified antibody, owing to the longer circulating half life vis-a-vis murine antibodies, an effective single treatment non-marrow ablative dosages of iodine-131 labeled chimeric antibodies range from between about 5 and about 40 mCi, more preferably less than about 30 mCI. Imaging criteria for, e.g., the 111 ln label, are typically less than about 5 mCI.
While a great deal of clinica I expérience has been galned with 1311 and .90Y, other radiolabels are known in the art and hâve been used for similar purposes. Still other radioisotopes are used for imaging. For example, additional radioisotopes which are compatible with the scope of the instant invention include, but are not limited to, 1231,1251,32P, 57Co, 64Cu, 67Cu, 77Br, 81Rb, 81Kr, 87Sr, 113ln, 127Cs, 129Cs, 1321,197Hg, 203Pb, 206BÎ, 177Lu, 186Re, 212Pb, 212Bi, 10 47Sc, 105Rh, 109Pd, 153Sm, 188Re, 199Au, 225Ac, 211A 213BL ln this respect alpha, gamma and beta emitters are ail compatible with in the instant invention. Further, ln view of the Instant disclosure it Is submitted that one skilled ln the art could readily détermine which radionuclides are compatible with a selected course of treatment without undue expérimentation. Tothis end, additional radionuclides which hâve already been used in clinîcal diagnosis include 1251,1231,
99Tc, 43K, 52Fe, 67Ga, 68Ga, as well as 111 ln. Antibodies hâve also been labeled with a variety of radionuclides for potential use ln targeted immunotherapy (Peirersz étal. Immunol. Cell Biol. 65: 111-125 (1987)). These radionuclides Include 188Re and 186Re as well as 199Au and 67Cu to a lesser extent. U.S. Pat No. 5,460,785 provides additional data regarding such radioisotopes and is incorporated herein by reference.
As previously discussed, the antibodies, or fragments thereof, of the Invention, can be administered ln a pharmaceutically effective amount for the ln vivo treatment of mammalian disorders. ln this regard, It will be appredated that the disclosed antibodies, or fragments thereof, will be formulated so as to fadlitate administration and promote stability of the active agent. Preferably, pharmaceutical compositions in accordance with the présent invention comprise a pharmaceutically acceptable, non-toxic, stérile carrier such as physiological saline, non-toxic buffers, preservatives and the like. For the purposes of the instant application, a pharmaceutically effective amount of a antibody of the invention, conjugated or unconjugated to a therapeutic agent, shall be held to mean an amount sufficient to achieve effective binding to a target and to achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder or to detect a substance or a cell. ln 30 the case of tumor cells, the polypeptide will be preferably be capable of interacting with selected immunoreactive antigens on neoplastic or immunoreactive cells and provide for an increase in the death of those cells. Of course, the pharmaceutical compositions ofthe présent invention may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the polypeptide.
In keeping with the scope of the présent disclosure, the antibodies of the invention may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect. The polypeptides ofthe invention can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody of the invention with a conventional pharmaceutically »
acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingrédient with which it is to be combined, the route of administration and other well-known variables. Those skilled ln the art will further appreciate that a cocktail comprising one or more species of polypeptides according to the présent Invention may prove to be particulariy effective.
VI. Methods of Treating Kallidin or des-Arg10-Kallidin-Associated Disease or Disorders
The anti-Kallidin or des-Arg10-Kallidin antibodies, or fragments thereof, of the Invention are useful for antagonizîng Kallidin or des-Arg10-Kallidin activity. Accordingly, in another aspect, the invention provides methods for treating Kallidin or des-Arg10-Kallidin-associated diseases or disorders by administering to a subject ln need of thereof a pharmaceutical composition comprising one or more anti-Kallidin or des-Arg10-Kallidin antibody, or antigen binding fragment thereof of the invention.
Kallidin or des-Arg10-Kallidin-associated diseases or disorders amenable to treatment Include, without limitation, pathophysiologic conditions such as inflammation, trauma, bums, shock, allergy, acute or chronic pain, and fibrosis, e.g., rénal fibrosis. ln certain exemplary, embodiments, antibodies of the invention may be issued to treat rénal fibrosis and associated acute kidney injury as well as chronic kidney diseases which are the main causes of end-stage renal failure.
One skilled in the art would be able, by routine expérimentation, to détermine what an effective, non-toxic amount of antibody (or additional therapeutic agent) would be for the purpose of treating a Kallidin ordes-Arg10-Kallidin-associated disease or disorder. For example, a therapeutically active amount of a polypeptide may vary according to factors such as the disease stage (e.g., stage I versus stage IV), âge, sex, medical complications (e.g., immunosuppressed conditions or diseases) and weight of the subject, and the ability of the antibody to elicit a desired response in the subject The dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. Generally, however, an effective dosage is expected to be in the range of about 0.05 to 100 milligrams per kilogram body weight per day and more preferably from about 0.5 to 10, milligrams per kilogram body weight per day.
VII. Exemplification
The présent invention is further illustrated by the following examples which should not be construed as further limiting. The contents of Sequence Listing, figures and ail référencés, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.
Furthermore, in accordance with the présent invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein ‘Sambrook et al., 1989); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic 5 Acid Hybridization [B.D. Hames & S.J.Higgins eds. (1985)]; Transcription And Translation [B.D.
Hames & S.J. Higgins, eds. (1984)]; Anima! Cell Culture [R.l. Freshney, ed. (1986)]; Immobilized
Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocole In MolecularBiology, John Wiley & Sons, Inc. (1994).
.4
Example 1: Hybridoma Production: Immunlzatlon of Mice with Kallidin Peptide Conjugated to KLH and Antibody Génération against Human BKR1 Ligands
The objective was to develop cross-reactive antibodies against Kallidin (KD; SEQ ID NO:1) and des-arg-Kallidin (DAKD; SEQ ID NO:2) that would inhibit these ligands binding to the human BKR1. Generally, Immunization of mice with KLH conjugated KD through additional cysteines on either the Ο or N- terminus of the peptide was used to obtain mouse splénocytes for fusion with mouse myeloma cell Unes as a fusion partner to produce the hybridomas.
Briefly, the Immunization protocol was as follows: BALB/c Mice (8-20 week-old naïve female) were immunized intraperitoneally with a mixture of even amounts of KLH-KD and KD-KLH ln phosphate buffered saline (PBS) as an antigen total of 100 ug per mouse mixed at 1:1 ratio of Sigma Adjuvant System (Sigma cat #6322) in a total volume of 200 μΙ per mouse (day 0). On day 21, mice were boosted with a mixture of even amounts of KLH-KD and KD-KLH ln PBS as an antigen total of 50 ug per mouse mixed at 1:1 ratio of Sigma Adjuvant System (Sigma cat #6322) in a total volume of 200 μΙ per mouse. On day 30, blood samples were harvested for KD spécifie antibody titer évaluation. On day 51, mice were boosted for fusion with a mixture of even amounts of KLH-KD and KD-KLH In PBS as an antigen total of 50 ug per mouse mixed at 1:1 ratio of Sigma Adjuvant System (Sigma cat #6322) in a total volume of 200 μΙ per mouse. At day 55 mice were sacrificed by
CO2 chamber, biood was coliected through the cardiac puncture and spleen was harvested for hybridoma production.
Hybridomas were made by fusing mouse myeloma cells that are déficient ln adenosine phosphoribosyltransferase (APRT) with spleen cells from mice immunized with spécifie antigens. A sélection System using HAT (hypoxanthine, azaserine, and thymidine) medium éliminâtes ail but the fusion cells that are APRT+. Successful hybridomas must also retain the immunoglobulin (Igh) heavy chain, one of the Immunoglobulin light chain loci and secrete functional antibody.
Hybridoma Production Medium (IMDM) was made by comblnlng the following: 500ml Iscove’s
Modified Dulbecco's Medium (HyClone SH30259.01), 50ml fêtai bovine sérum (HyClone SH30070.03), 5ml L-glutamine (Gibco Invitrogen cat# 25030), 5mi non-essential amino acids (Gibco Invitrogen cat # 11140050), 5mi sodium pyruvate (Gibco Invitrogen cat # 11360070), 5ml 0.1% penicillin-streptomycin (Gibco Invitrogen cat # 15140148). The medium was filtered before use. Expansion medium was made by combining the following: 1000ml sérum free medium (Gibco
Hybridoma SFM # 12045), 100ml 10% HyClone SuperLow IgG Defined FBS # SH30898.03 and
10ml penicillîn/streptomycin. Freezing medium was 45ml heat Inactivated FBS (HyClone
SH30070.03) and 5ml DMSO, filter sterilized. Other materials Included the following : HAT (50x) was obtained from Sigma-Aldrich (# HO262); Hybridoma Fusion and Cloning Supplément (50X) (Roche Diagnostics 11 363 735 001); Trypan Blue S ta in 0.4% (Invitrogen cat # 15250-061 or t
T10282); PEG 1500 in 75 mM Hepes 50% w/v (Roche cat# 783641(10783641001). Ail the reagents except HAT and Hybridoma Fusion and doning supplément were used at 37°C.
Table 2. Peptide Reagents Used in Immunlzatlon and Screening
Peptide No. SEQ ID ND. Peptide Sequence Peptide Name Alternative Name
1 5 RPPGFSPFR bradykinin BK
2 117 biotin-RPPGFSPFR b-BK
3 71 RPPGFSPFR-biotin BK-b
4 72 KLH-RPPGFSPFR KLH-BK
5 73 RPPGFSPFR-KLH BK-KLH
6 1 KRPPGFSPFR kallidin KD
7 74 biotin-KRPPGFSPFR b-KD
8 75 KRPPGFSPFR-bîotin KD-b
9 76 KLH-KRPPGFSPFR KLH-KD
10 77 KRPPGFSPFR-KLH KD-KLH
11 6 RPPGFSPF desArg9bradykinin DABK
12 78 biotin-RPPGFSPF b-DABK
13 79 RPPGFSPF-biotin DABK-b
14 80 KLH-RPPGFSPF KLHDABK
15 81 RPPGFSPF-KLH DABK-KLH
16 2 KRPPGFSPF desArqlOkallidin DAKD
17 82 biotin-KRPPGFSPF b-DAKD
18 83 KRPPGFSPF-biotin DAKD-b
19 84 KLH-KRPPGFSPF KLH-DAKD
20 85 KRPPGFSPF-KLH DAKD-KLH
21 3 RRPPGFSPFR Kallidin like peptide KLP
22 86 biotin-RRPPGFSPFR b-KLP
23 87 RRPPGFSPFR-biotin KLP-b
24 88 KLH-RRPPGFSPFR KLH-KLP
25 89 RRPPGFSPFR-KLH KLP-KLH
26 90 RRPPGFSPF desArgIOkallidin like peptide DAKLP
27 91 biotin-RRPPGFSPF b-DAKLP
28 92 RRPPGFSPF-biotin DAKLP-b
29 93 KLH-RRPPGFSPF KLH-DAKLP
30 94 RRPPGFSPF-KLH DAKLP-b
31 95 RPPGF bradykinln1-5 BK15
32 96 biotin-RPPGF b-BK15
Briefly, three or four days before the fusion, the mouse was boosted with an antigen of Interest either Intraperitonealy or intravenously. On the day of the fusion, the mouse was sacrificed In CO2 chamber, blood was coliected by cardiac puncture and the spleen was taken out and placed Into 10 ml of sérum free IMDM in a Pétri dish. Fusion partner cells myeloma: FO (ATCC ref CRL-1646)/
X63 Ag8.653 (ATCC ref CRL1580) were grown at a log phase, then split one day before the fusion (1:2 and 1:5), and collected into 20 ml centrifuge tubes, spun and resuspended the pellet in 10ml IMDM. The pellet was washed two times with sérum free IMDM medium. Ail the centrifugations are performed at 1570 rpm for 5 min. Final resuspension was in 10ml sérum free IMDM. The connective tissue was dissected away from the spleen. The spleen was injected with 1 ml of sérum free IMDM preheated to 37° C by 1 ml syringe and 25-gauge needle. Splénocytes are squeezed out of the fibroelastic coat by forceps and washed two times In 10 ml of sérum free IMDM (including initial spin) and were resuspended in 10ml sérum free IMDM. Cells were counted in Countess Automated Cell Counter.
Fusion partner cells and splénocytes were combined In one 50ml tube at ratio of 1:2 to 1:10 (by cell number) and spun down at 970 rpm for 10 min (slow spin) to form a loose pellet. After the slow* spin, supematant was taken out with the précaution not to disturb the pellet, but minimize the amount of liquid over the cells in order not to dilute PEG 1500. The last remaining medium was reserved and added back after the PEG is added (below). Preheated PEG 1500 (37°C, total 1 ml) was added drop by drop to the cell pellet over 1 minute period of time and cells were mixed after every drop of PEG was added. Pellet was incubated with PEG for another 1 minute followed by addition of 10 ml of serum-free IMDM medium over 1 minute, so that the first 1 ml out of 10 is added over 30 sec. Cells underwent slow spin at 970 rpm for 10 min and supematant decanted.
Into (2) 100ml troughs, the following was added: 70ml IMDM with 10% FBS, 2ml HATand 2ml Hybridoma and Fusion Cloning Supplément. Cells were résuspended in 10ml IMDM with 10% FBS and split into (2) 50ml tubes (5ml cells/tube) and 25ml IMDM with 10% FBS was added. The resulting 30ml was transferred to the troughs containing 70ml HBSS/HAT/cloning supplément and 200ul cells/well were pipetted into (10) 96-well plates. Fusion was ready for screening by ELISA (50ul) about 10 to 14 days later, or when medium in the wells tums yellow. After the primary screening, positive clones are selected, numbered and moved to a 24-well plate in 500 ul per well of IMDM with 10% FBSHI. Hybridoma supematants were screened by ELISA on streptavidin plated coated with N- and C-term biotinylated peptides (see below).
Example 2: Characterlzatlon and Sélection of Hybrldomas Expressing Antibodies Against
Human BKR1 Ligands
Hybridoma supematants were screened by ELISA on streptavidin plated coated with N- and C-term biotinylated peptides (see e.g., those set forth in Table 2) and then antibody binding kinetics were determined for confirmed positive hybridoma clones.
The abîlity of the antibodies In hybridoma supematants to bind to BKR1 ligand peptide was evaluated with an ELISA assay. DAKD-biotin or KD-biotin peptides was coated on a 96-well SA plate in phosphate buffered saline (PBS) buffer for an hour at room température at 5 ug/ml, and the nonspecific binding sites were blocked with 1% bovine sérum albumin (BSA) in PBS buffer. This plate was used to perform primary and secondary screening ofthe crude hybridoma supematants.
Hybridoma supematants were added to the plates for binding to the coated KD or DAKD peptides. After 1 hour Incubation, the plate was washed and bound antibodies were detected using horseradish peroxidase (HR P) conjugated secondary antibody (HRP-goat anti-mou se IgG (H+L): Jackson ImmunoResearch Labs # 115-035-166) and developed using 2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic add) (ABTS) substrate (Roche diagnostics #11 204 521 001). Data was analyzed using Excel. The antibodies showing positive signais (2 fold higher than 1:10000 sérum dilution ELISA signal) were selected and re-screened In duplicates for confirmation. Confirmed positive hybridoma clones were selected and subjected to binding dissodation rate ranklng by Blacore.
For antibody binding kinetics, the instruments used were the BIACORE 2000 or BIACORE 3000 (GE Heaithcare), designed for biomolecular interaction analysis (BIA) In real time. The sensor chip used was SA chip (GE Heaithcare) with streptavidin covalently immobilized on a carboxymethytated dextran matrix. Each sensor chip has four parallel flow cells (Fc). Every biotinytated BKR1 or BKR2 ligand peptides were immobilized to one of the flow cells 2 to 4 (Fc2 to Fc4) In the SA chip for binding dissodation rate screening and selectivity screening. Flow cell 1 (Fc1) was reserved and Immobilized with a random peptide (biotinylated at one terminus) with equal or close peptide length in comparison to the testing ligand peptides as the négative control. In screening assays, cell culture supematants of the hybridoma dones selected through primary screening or of transiently expressed humanized variants were Injected over Immobilized peptides, Hybridoma cell culture media was also Injected over the chip surface as blank to establish a baseline. After subtracting signais of Fc1 and blank buffer runs, the dissociation rate of the antibodies from the supematants to each peptide was analyzed and ranked using BIAevaluation software. Only the antibody clones that demonstrated superior (kd < 10 -4 1/s) binding dissodation rate were selected for subcloning and further characterization. In kinetics analysis, the corresponding biotin-peptides Identified in screening for the testing antibody were immobilized In Fc2 to Fc4 while Fc1 with a random peptide used as référencé cell. Each purified antibody selected from screenings were made Into a sériés of two fold dilutions in running buffer (1 x HBSEP buffer, GE Heaithcare) between 0.1 to 10 nM. Binding assodation rate, dissodation rate and the overall affinity were calculated in BIAevaluation. Antibody binding kinetics for each antibody was always confirmed in triplicate assays using Biacore.
A total of 8 mice were Immunized with mixed KLH-KD/KD-KLH and KLH-DAKD/DAKD-KLH and the spleens were fused using the above protocols. After primary screening of about 7680 hybridoma clones in ELISA with DAKD-blotin and KD-biotin, only 76 clones were confirmed positive and selected for binding dissodation rate ranklng In Biacore 3000/2000 over the immobilized DAKDbiotin and KD-biotin on Streptavidin (SA) chips. Among those, 8 hybridoma clones with binding dissodation rate <=of 104 were subcloned, sequenced, purified and further characterized (see
Table 3).
Table 3 : Immunlzation Results with KLH-KD/KD-KLH and KLH-DAKD/DAKD-KLH
Clone ID
Ligand Peptide Used In Assay Assay B21 C63 F151 F306 12 I8 122“ 154“
DAKD b-DAKD ELISA + + • • + + + +
Biacore (KD.M) NS NS
DAKD-b ELISA + + + + + + + +
Biacore (KD.M) 4.15 E-11 1.42 E-tO 1.60 E-10 1.60 E-10 1.10 E-10 6.25 E-10 NTD NTD
DAKD (50nM) FLIPR (nM) 22- 25. 9 6.9 6.9 9.4 8.1
DABK b-DABK ELISA +/- +/- • * + + + +
Biacore (KD.M) NS NS
DABK-b ELISA + +/- + + + +
Biacore (KD.M) • NS NS
DABK FLIPR (nM)
* m A m ELISA *
Biacore (KD,M) NS NS
BK-b ELISA + + + + +
Biacore (KD,M) 8.57 E-09 NS NS
si m FUPR (nM)
Q b-KD ELISA +
Biacore (KD,M) NS NS
KD-b ELISA + + + + + + + +
Biacore (KD,M) 8.4 E-11 2.21 E-10 2.9 E-11 2.9 E-11 7.8 E-11 7.53 E-10 NTD NTD
KD (15nM)M) FLIPR (nM) 12 3.0 3.0 7.3 8.3 25 30
NA = not applicable, négative in ELISA NS = nonspecific binding NTD = not to be determined -* = residual binding (low RU in Biacore)
Based on results seen in Table 3, five clones with unique sequences were selected for kinetic studies. These antibodies were highly sélective for DAKD-biotin, KD-biotin, DAKLP-biotin and KLP17139 biotin binding (see Table 4). They do not bind to other kinin peptides or to peptides biotinylated at the N-terminus.
Table 4. Summary of Klnetlcs of Selected anti-DAKD/KD Antibody Candidates
DAKD-b KD-b
Antibody koff KD koff KD
C63 9.36E-05 1.42E-10 1.00E-04 2.21 E-10
B21 9.89E-05 4.15E-11 2.04 E-04 8.40E-11
F151 1.36E-04 1.62E-10 2.00E-05 2.88E-11
122 3.19E-04 2.17E-10 2.10E-05 4.40E-12
154 3.06E-05 9.53 E-12 3.88E-05 1.12E-11
DAKLP-b KLP-b
koff KD koff KD
C63 n/b n/b n/b n/b
B21 2.30E-04 1.34E-10 1.12E-04 1.92E-10
F151 6.58E-05 2.12E-10 S1.0E-06 1.66E-11
122 S1.0E-06 •1.83E-12 1.03ΕΌ5 1.82E-12
154 5.66E-05 1.17E-11 6.04E-05 9.56E-12
n/b = no binding
Additional immunization were performed with an array of immtinogens (see list of peptides, Table
2) for generating antibodies blocking the rodent BKR1 ligands, DABK and DAKD as well as antibodies with other binding specificities against different member of kinin family of peptides.
Table 5 lists the heavy and light sequences of the antibodies generated.
Table 5. Heavy and Light Chain Sequences of Antibodies
Antibody Isotype SEQ ID NO. Heavy Chain Sequence
B21 £ O 97 LPEFOVKLEESGAELVRSGASVKLSCTASGFNIKDYYLHWVKQRPEOGLEWIGWI DPENGDTGYARKFQGKATHTADTSSNTVYLHLSSLTSEDTAVYYFNAWEYDGYYD
LDYWGOGTSVTVSSAKTTPPSVYGSS
C63 98 LP EFOVOLEESGGGLVOPGGSMKLS CVAS G FT FSNYWMNWVRQS PEKGLEWVAE IRSKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYÇIGEDYG
GDYWGOGTSVTVSSAKTTPPSVYGSS
F151 99 LPEFEVOLEESGPELVKPGTSVKVSCKASGYSFTDYNIYWVKQSHGKSLEWIGYF DPYNGNTGYNQKFRGKATLTVDKSSSTAFMHLSSLTSDDSAVYYÇANYYRYDDHA
MDYWGQGTSVTVS SΑΚΤΊ P P SVYGS S
Ι22 100 LPEFEVKLOESGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEOGLEWIGWV DPENGDSDYAPKFQGKATMTADTSSNTVYLQFSSLTSEDTAVYYÇNAFEYDGNYS
SLDFWGQGTSVTVSSAKTTPPSVYGSS
154 101 LPEFEVKLEOSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEOGLEWIGWV DPENGDSDYAPKFQGKATMTADTSSNTVYLQFSSLTSEDTAVYYCNAFEYDGNYS
PLDFWGQGTSVTVSSAKTTPPSVYGSS
B21 * 0 CD E 118 EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIGWIDPEN GDTGYARKFQGKATMTADTSSNTVYLHLSSLTSEDTAVYYFNAWEYDGYYDLDYW
GQGTSVTVSSAKTTPPS
C63 119 EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRSKS NNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEOTGIYYÇIGEDYGGDYWGQ GTSVTVSSAKTTPPS
F151 120 EIQLOOSGPELVKPGTSVKVSCKASGYSFTDYNIYWVKQSHGKSLEWIGYFDPYN gntgynqkfrgkatltvdkssstafmhlssltsddsavyyçanyyryodhamdyw
GQGTSVTVSSAKTTPPS
I22 121 EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEOGLEWIGWVDPEN GDSDYAPKFQGKATMTADTSSNTVYLQFSSLTSEDTAVYYÇNAFEYDGNYSPLDF
WGQGTSVTVSSAKTTPPS
154 122 EVOLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQGLEWIGWVDPEN GDSDYAPKFQGKATMTADTSSNTVYLQFSSLTSEDTAVYYCNAFEYDGNYSPLDF WGQGTSVTVS SAKTT P PS
Antibody Isotype SEQ ID NO. LlghtChainSequence
B21 « 0 102 ELDIVMTQTTLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLI YLVSKLDSGVPDRFTGSGSGTDFTLKIIRVEAEDLGVYYÇLQGTHFPYTFGGGTK LEIKRADAAPTVSIFPPSKLELY
C63 103 ELDIVLTQSPSSLAVSVGEKVTMSÇKSSQSLLYSSDQRNYLAWYQQRSGQSPKLL IYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGT KLEIKRADAAPTVSIFPPSKLELY
F151 104 eldivmtqtpsslavsvgekvtmsckssqsllytsnqknylawyqqkpgqspkpl IYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAIYYCOQYYSYPWTFGGGT KLEIKRADAAPTVSIFPPSKLELY
I22 105 ELDIVITQTTLSLSVPIGQPASISÇKSRQSLLYSNGETYLNWLLQRPGQSPKRLI YLVSKLDSGVPDRFTGSRSGTDFTLKISRVESEDLGVYYCMQGTHFPYTFGGGTK LEIKRADAAPTVSIFPPSKLE LY
154 106 ELDIVITQSTLTLSVPIGQPASISÇKSSQSLLYSNGETYLNWLLQRPGOSPKROI YLVSKLDSGVPDRFTGSRSGTDFTLKISRVESEDLGVYYCMQGTHFPYTFGGGTK LEIKRADAAPTVSIFPPSKLELY
B21 mlgG1/K 123 DWMTQT P LT LS VTIGQPASISÇKS SQSLL YS NG KTY LNWLLQRPGQS PKRLIYL VSKLDSGVPDRFTGSGSGTDFTLKIIRVEAEDLGVYYÇLQGTHFPYTFGGGTKLE IKRADAAPT
C63 124 DIVMSOSPSSLAVSVGEKVTMSCKSSQSLLYSSDQRNYLAWYQQRSGQSPKLLIY WASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKL EIKRADAAPT
F151 125 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKPLIY WASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAIYYÇQQYYSYPWTFGGGTKL EIKRADAAPT
I22 126 DIVMTOSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIY GASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYÇQNDHSYPLTFGAGTKL ELKRADAAPT
154 131 DWMTOT PLTLS V PIGOPASISCKSSQSLLYS NGET YLNWLLQRPGQS PKRLIY L VSKLDSGVPDRFTGSRSGTDFTLKISRVESEDLGVYYCMQGTHFPYTFGGGTKLE IKRADAAPT
Single underscore = CDR région; double underscore - signature amino acids for Identifying CDRs
EXAMPLE 3: Génération of Surrogate Antibody for Murine Animai Studies
A surrogate antibody to be used in murine animai studies needed to be able to bind and neutralize rodent BKR1 ligands, DABK and DAKLP (mouse équivalent of DAKD). In order to generate the required surrogate antibody, mice were first immunized with DABK and/or DAKD with KLH directly conjugated to the N-terminais of the peptides. Biotin-DABK/biotin-DAKD (biotinylation directly on
N-terminus ofthe peptide) positive hybridoma clones from ELISA screening were selected for scaling up and purification. The antibodies listed in Family 7 (see Table 12) that demonstrated high binding affinities to biotin-DABK, biotin-DAKLP and blotin-DAKD were selected based on Biacore
I direct binding assay (Table 10). However, these Family 7 antibodies showed no binding to the native, unmodffîed DABK and DAKD peptides in compétitive ELIS A, and lacked neutralizing functionality ln a calcium influx assay with Functional Drug Screening System (FDSS) (Hamamatsu Photo nies K.K., Japan), Moreover, the biotin-DABK and biotin-DAKD completely lost bioactivity ln the FDSS assay in comparison to the native, unmodified DABK and DAKD peptides (data not shown).
It was hypothesized that the direct N-terminus conjugation of KLH and biotin prevented the native confirmation of DABK and DAKD to form. With the aim to restore the native conformation in KLH10 and biotin- conjugated peptides, linkers were designed and added to the N-terminus of DABK and/or DAKD with the intention to cushion the KLH/biotin conjugation effects on peptide conformation. Poly-glycine linkers were first attempted and tested because of their simple, nonpolar and neutral properties based on modeling results. The FDSS assay results indicated that the gly-gly-gly (3G) linker was the best according to its ability to restore the bîoactivities of KLH and 15 biotin conjugated DABK and DAKD peptides (data not shown). Therefore, KLH-3G-DABK was chosen to immunize mice. And btotin-3G-DABK and biotin-3G*KD were used in binding based screening assays (ELISA and Biacore). Several DABK/DAKD spécifie antibodies (Family 3, see Table 13) were Identified in this new round of surrogate antibody hybridoma sélection. EE1 was selected as the lead surrogate antibody based on its superior binding afïinity and neutralization 20 activity against native DABK/DAKD and lack of cross-reactivity to other peptides ( see Tables 6 12)
Antibodies with different specificities were generated when using the different immunogens listed ln Table 13. Family 4 antibodies were spécifie to the BKR2 receptor ligands, BK and KD. Family 5 25 antibodies specifically bind to the C terminus of BK and DABK. Family 6 antibodies bind BK, DABK and DAKD but do not bind to KD.
Additional linkers were evaluated for binding to the surrogate EE1 antibody for their ability to fit Into the DABK/DAKD binding pocket in EE1, including longer poly-glycine linkers, poly-alanine linkers 30 and preexisting linkers such as polyethylene glycol (PEG2) linker and aminohexanoic acid (Ahx) linker (a 6-carbon inert linker). Ail linker peptides were custom synthesized by Abgent (Can Diego, CA). Ail tested biotinytated peptides with linkers (biotin-linker-DABK/DAKD) bound well to EE1, indicating that any inert N-terminus linkers helped DABK and DAKD peptides to retain their native bioactive conformation when conjugated with biotin and other molécules, ln contrast, no binding or 35 poor binding to EE1 was observed with biotin-DABK and biotin-DAKD, peptides that hâve direct Nterminal biotin conjugation (see Figure 1).
The binding klnetics of generated antibodies are summarized in Tables 5-11. Then, ail antibodies generated were sorted into families and their binding specificities are summarized below in Table
12. Table 13 provides the heavy and light chain sequences of antibodies that were placed into family 1 and family 2 based on their binding specificity (see Table 12).
Table 6. Summary of Antibody Klnetlcs to b-3G-DABK and b-3G-DAKD Peptides
Clone b-3G-DABK b-3G-DAKD
Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M)
DD20 1.5E+06 2.3E-04 1.6E-10 4.7E+05 4.0E-04 8.8E-10
UR11 2.0E+05 3.0E-04 1.5E-09 3.0E+05 1.6E-03 5.2E-O9
DD7 2.3E+05 6.0E-04 2.7E-09 2.1E+05 1.4E-03 6.6E-09
EE1 4.4E+05 1.2E-04 2.8E-10 4.4E+05 2.0E-04 4.5E-10
EE36 4.3E+03 5.3E-04 1.2E-07 n/b n/b n/b
UR29 n/b* n/b n/b n/b n/b n/b
JK3 3.44E+05 3.91 E-05 1.14E-10 3.18E+05 5.07E-05 1.60E-10
LR4 n/b n/b n/b n/b n/b n/b
LR16 n/b n/b n/b n/b n/b n/b
LR6 n/b n/b n/b n/b n/b n/b
LR12 n/b n/b n/b n/b n/b n/b
NR1 n/b n/b n/b n/b n/b n/b
NR15 n/b n/b n/b n/b n/b n/b
n/b* = non-specific binding, n/b = no binding
Table 7. Summary of Antibody Klnetlcs to b-3G-DAKLP and b-3G-BK Peptides
Clone b-3G-DAKLP b-3G-BK
Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M)
DD20 3.1E+05 6.1E-04 2.0ΕΌ9 3.0E+05 7.1E-04 2.3ΕΌ9
UR11 n/b n/b n/b n/b n/b n/b
DD7 1.7E+05 2.1E-03 1.2E-08 1.3E+05 8.8E-04 6.8E-09
EE1 4.2E+05 2.9E-04 6.8E-10 2.5E+05 2.6E-03 1.1E-08
EE36 n/b n/b n/b n/b n/b n/b
UR29 n/b n/b n/b n/b n/b n/b
JK3 ND ND ND n/b n/b n/b
LR4 n/b n/b n/b n/b n/b n/b
LR16 n/b n/b n/b n/b n/b n/b
LR6 n/b n/b n/b n/b n/b n/b
LR12 n/b n/b n/b n/b n/b n/b
NR1 n/b n/b n/b n/b n/b n/b
NR16 n/b n/b n/b n/b n/b n/b
n/b = no binding: ND = not determined
Table 8. Summary of Antibody Klnetlcs to b-3G-KLP and b-3G-KD peptides
Clone b-3G-KLP b-3G-KD
Ka(1/Ms) Kd(1/s) KD(M) Ka(l/Ms) Kd(1/s) KD(M)
DD20 3.8E+05 5.7E-04 1.5E-09 n/b n/b n/b
UR11 n/b n/b n/b 1.2E+05 1.3E-03 1.1E-08
DD7 1.5E+05 2.1E-03 1.5E-08 2.2E+05 1.8E-03 8.4E-09
EE1 4.0E+05 2.1E-03 5.3E-09 n/b n/b n/b
EE36 n/b n/b n/b n/b n/b n/b
UR29 n/b n/b n/b n/b n/b n/b
JK3 ND ND ND n/b n/b n/b
LR4 n/b n/b n/b n/b n/b n/b
LR16 n/b n/b n/b n/b n/b n/b
LR6 n/b n/b n/b n/b n/b n/b
LR12 n/b n/b n/b n/b n/b n/b
NR1 n/b n/b n/b n/b n/b n/b
NR15 n/b n/b n/b n/b n/b n/b
n/b = no binding; ND = not determined
Table 9. Summary of Antibody Klnetlcs to DABK-b and DAKLP-b Peptides
Clone DABK-b DAKLP-b
Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M)
DD20 n/b n/b n/b n/b n/b n/b
UR11 n/b n/b n/b n/b n/b n/b
DD7 n/b n/b n/b n/b n/b n/b
EE1 n/b n/b n/b n/b n/b n/b
EE36 n/b n/b n/b n/b n/b n/b
UR29 1.5E+06 5.8E-05 3.9E-11 3.0E+06 2.1E-03 6.8E-10
JK3 n/b n/b n/b n/b n/b n/b
LR4 n/b n/b n/b n/b n/b n/b
LR16 n/b n/b n/b n/b n/b n/b
LR6 n/b n/b n/b n/b n/b n/b
LR12 n/b n/b n/b n/b n/b n/b
NR1 n/b n/b n/b n/b n/b n/b
NR15 n/b n/b n/b n/b n/b n/b
n/b = no binding; ND = not determined
Table 10. Sum mary of Antibody Kinetics to BK-b and b-BK Peptides
Clone BK-b b-BK
Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M)
DD20 n/b n/b n/b n/b n/b n/b
UR11 n/b n/b n/b n/b n/b n/b
DD7 n/b n/b n/b n/b n/b n/b
EE1 n/b n/b n/b n/b n/b n/b
EE36 n/b n/b n/b n/b n/b n/b
UR29 1.5E+06 1.0E-04 7.2E-11 n/b n/b n/b
JK3 n/b n/b n/b n/b n/b n/b
LR4 n/b n/b n/b n/b n/b n/b
LR16 n/b n/b n/b n/b n/b n/b
LR6 n/b n/b n/b n/b n/b n/b
LR12 n/b n/b n/b n/b n/b n/b
NR1 n/b n/b n/b 8.69E+04 8.77E-04 1.01 E-08
NR15 n/b n/b n/b 2.95E+05 1.09E-03 3.68E-09
n/b = no binding; ND = not determined
Table 11. Summary of Antibody Klnetlcs to b-DABK and b-DAKD Peptides
Clone b-DABK b-DAKD
Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M)
DD20 n/b n/b n/b n/b n/b n/b
UR11 n/b n/b n/b n/b n/b n/b
DD7 n/b n/b n/b n/b n/b n/b
EE1 n/b n/b n/b n/b n/b n/b
EE36 n/b n/b n/b n/b n/b n/b
UR29 n/b n/b n/b n/b n/b n/b
JK3 n/b n/b n/b n/b n/b n/b
LR4 1.48E+05 1.04E-03 7.15E-09 3.27E+05 7.63E-04 2.36E-09
LR16 4.34E+05 4.38E-O5 1.01 E-10 2.07E+05 3.39E-03 1.65E-O8
LR6 n/b n/b n/b n/b n/b n/b
LR12 2.91 E+05 5.40E-04 3.63E-09 n/b n/b n/b
NR1 n/b n/b n/b n/b n/b n/b
NR15 n/b n/b n/b n/b n/b n/b
n/b = no binding; ND = not determined
Table 12. Summary of Antibody Klnetlcs to b-DAKLP and b-KD Peptides
Clone b-DAKLP b-KD
Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M)
DD20 n/b n/b n/b n/b n/b n/b
UR11 n/b n/b n/b n/b n/b n/b
DD7 n/b n/b n/b n/b n/b n/b
EE1 n/b n/b n/b n/b n/b n/b
EE36 n/b n/b n/b n/b n/b n/b
UR29 n/b n/b n/b n/b n/b n/b
JK3 n/b n/b n/b n/b n/b n/b
LR4 1.84E+05 2.58E-04 1.40E-09 n/b n/b n/b
LR16 2.34E+05 1.11E-04 4.74E-10 n/b n/b n/b
LR6 6.80E+05 4.01 E-04 7.45E-10 n/b n/b n/b
LR12 n/b n/b n/b n/b n/b n/b
NR1 n/b n/b n/b 1.66E+05 5.81E-03 3.56E-08
NR15 n/b n/b n/b 7.66E+05 5.66E-03 7.41 E-09
n/b = no binding; ND = not determined
Table 13. Summary of Antl-klnln peptide antibody génération
Antibody Families Immunogens Représentative antibodies Binding Speclficlty
Family 1 KD-KLH + KLH-KD or DAKD-KLH + KLHDAKD F151, B21,122,154 N-terminus of DAKD (DAKLP) and KD (KLP)
Family 2 KD-KLH + KLH-KD or DAKD-KLH + KLHDAKD C63 N-terminus of DAKD and KD
Family 3 KLH-3G-DABK EE1,DD20, JK3 C-terminus of DAB K and DAKD (DAKLP)
Family 4 KLH-BK and BSA-BK NR15, NR1 C-terminus of BK and KD
Family 5 KLH-BK UR29 N-terminus of BK and DABK
Family 6 KLH-BK UR11 BK, DABK and DAKD
Family 7 KLH-DABK or KLHDAKD LR4, LR6, LR12 and LR16 no binding with native peptides
Example 4: Characterizatlon of des-arg-KInln Ligand Déplétion using Calcium Mobllizatlon
A functional assay was used to further characterize the seven families of generated antibodies. The Bradykinin B1 Receptor signaling Is Gq coupled, therefore receptor activation can be monitored using Gq activation of IP3 and downstream calcium mobilization. HEK mBKR1 (recombinant mouse bradykinin B1 receptor) cells or MRC5 (endogenous expression of bradykinin B2 receptors; (ATCC CCL-171)) were used to measure calcium mobilization.
Briefly, the mouse Bdkrbl gene (sequence provided below) was amplified from mouse lung cDNA (Biochain, Cat# C1334152) using PCR primers 804_cGWY_F: 5’-AAAAGCAGGCTTAGGAGCGGCCGCCATGGCGTCCCAGGCCTCGCTG-3‘ (SEQ ID NO: 107) and 804_cGWY_R: S’-CAAGAAAGCTGGGTCGGATCCTTATAAAGTTCCCAGAACCCTGGTC-S' (SEQ ID NO: 108) and Pfu Polymerase (Agitent Technologies, Cat# 600264) and cloned into pDONR201 using BP donase enzyme mix (Invitrogen, Cat# 11789-020). In parallel, the pEAK8 expression vector (EDGE Biosystems) was modified by inserting a N-terminal HA tag (GCATACCCATACGACGTCCCAGACTACGCT, GenBank SEQ ID NO:109 CY100443) into pEAK8 iinearized with EcoRI and Hindlli (vector pEAK8-nHA) and subséquent insertion ofthe Gateway cassette B (invitrogen, Cat# 11828-029) into pEAK8_nHA digested with EcoRI and Notl and bluntended with Klenow polymerase (NEB, cat# M0210S) resulting in vector pEAK8_nHA_DEST. Next mouse Bdkrbl was subcloned into pEAK8_nHA_DEST using LR clonase (Invitrogen, Cat# 1179117139
100). 293-PSC ceîls were then transfected with pEAK8-Bdkrb1 plasmid using Fugene 6 transfection reagent The cells were put under antibiotic (puromycin) sélection 24 hours after transfection, and sélection was maintained to generate a stable cell line. Presence ofthe Bdkrbl gene in the résultant stable cell lines was confirmed using real time RT-PCR, and by agarose gel 5 electrophoresis. Cell surface expression of the Bradykinin B1 receptor was performed by using an antibody against the N-terminal-HA tag (Covance, Cat# MMS-101P) on the Bradykinin B1R on a FACS instrument Functional activity ofthe Bradykinin B1 receptor was demonstrated In calcium mobilization assay with sélective agonlsts.
Bdkrbl gene subcloned into cells:
ATGGCGTCCCAGGCCTCGCTGAAGCTACAGCCTTCTAACCAAAGCCAGCAGGCCCCTCCCAACATCACCTCCT GCGAGGGCGCCCCGGAAGCCTGGGATCTGCTGTGTCGGGTGCTGCCAGGGTTTGTCATCACTGTCTGTTTCTT TGGCCTCCTGGGGAACCTTTTAGTCCTGTCCTTCTTCCTTTTGCCTTGGCGACGATGGTGGCAGCAGCGGCGG CAGCGCCTAACCATAGCAGAAATCTACCTGGCTAACTTGGCAGCTTCTGATCTGGTGTTTGTGCTGGGCCTGC 15 CCTTCTGGGCAGAGAACGTTGGGAACCGTTTCAACTGGCCCTTTGGAAGTGACCTCTGCCGGGTGGTCAGCGG
GGTCATCAAGGCCAACCTGTTCATCAGCATCTTCCTGGTGGTGGCCATCAGTCAGGACCGCTACAGGTTGCTG GTATACCCCATGACCAGCTGGGGGAACCGGCGGCGACGGCAAGCCCAAGTGACCTGCCTGCTCATCTGGGTAG CTGGGGGCCTCTTGAGCACCCCCACGTTCCTTCTGCGTTCCGTCAAAGTCGTCCCTGATCTGAACATCTCTGC CTGCATCCTGCTTTTCCCCCACGAAGCTTGGCACTTTGTAAGGATGGTGGAGTTGAACGTTTTGGGTTTCCTC 20 CTCCCATTGGCTGCCATCCTCTACTTCAACTTTCACATCCTGGCCTCCCTGAGAGGACAGAAGGAGGCCAGCA
GAACCCGGTGTGGGGGACCCAAGGACAGCAAGACAATGGGGCTGATCCTCACACTGGTAGCCTCCTTCCTGGT CTGCTGGGCCCCTTACCACTTCTTTGCCTTCCTGGATTTCCTGGTCCAGGTGAGAGTGATCCAGGACTGCTTC TGGAAGGAGCTCACAGACCTGGGCCTGCAGCTGGCCAACTTCTTTGCTTTTGTCAACAGCTGCCTGAACCCAC TGATTTATGTCTTTGCAGGCCGGCTCTTTAAGACCAGGGTTCTGGGAACTTTATAA (GenBank
NM_007539; SEQ ID NO:110>
HEK mBKR1 or MRC5 cells were plated into 384 well clear bottom plates in growth medium, and allowed to attach overnight. Then growth media was removed, cells were washed in assay buffer 30 (HBSS, 20mM HEPES, 2.5 mM probenedd), then dye-loaded with 0.5 uM Fluo-4AM, a cell permeable calcium sensing dye, with 0.04% Pluronic Acid for 1 hr at 37C. The AM ester Is cleaved, and the calcium dye is retained in the cytoplasm. After 1 hr, the cells were washed to remove excess dye, and 20ul of residual buffer remalned on the cells. Treatments were added as 2x solutions on the Functional Drug Screening System from Hamamatsu (FDSS), and the calcium 35 mobilization was monitored kinetically for at least 4 minutes. B1R or B2R receptor activation results in Galpha q mediated activation of phospholipase C and IP3 mediated calcium mobilization. The Fiuo-4 dye chelates the released calcium, and a robust change In fluorescence is observed. The results were exported as max-min relative fluorescence units to normalize for différences between cell density or dye loading across the plate.
Ligand potency was determined each day by running concentration response curves of iigand, and an approximate EC70-80 concentration of ligand was selected for incubation with antibodies. An EC80 concentration was selected because it is on the linear range of the détection curve and there was ample window to see a decrease with antagonists or ligand depleting antibodies. Dose 45 response curve of antibodies were allowed to bind a EC80 concentration iigand, and the extent of
I ligand déplétion was monitored using change in fluorescence. Results were normalized to buffer and EC80 ligand response, and an EC50 for ligand déplétion was calculated. The results were then reported as molar ratio which corresponds to the Antibody concentration that reduces depletes
50% of the ligand response (i.e., EC50 of Ab) divided by the ligand concentration used. The theoretical max should be 0.5 because one unit of antibody should be able to deplete 2 units of ligand, but we hâve seen lower values in practice but that may be a reflection of the insensitivity of the détection method for low ligand concentrations, rather than a stochiometric constraint for the antibody. The results of these experiments are set forth in Tables 14-16.
Ail family 1 and family 2 antibodies (see Table 13) demonstrated superior binding kinetics by Biacore (Table 3) and neutralization activity as measured by calcium mobilization against DAKD and KD peptides (Tables 14 and 15). The antibodies were further analyzed for their thermal stability and sequence suitability for humanization. F151 was advanced for humanization because itwas thermally stable, there were no problematic residues in the CDR régions and it was cross-reactive to the mouse ligand KLP and DAKLP.
Table 14: Characterizatlon of des-arg-KInln Ligand Déplétion using Calcium Mobilization in HEK mBKR1 cells
Déplétion of DAB K Déplétion of DAKD Déplétion of DAKLP
Family Antibody DABK (Mean Molar Ratio) SD Molar Ratio n DAKD (Mean Molar Ratio) SD Molar Ratio n DAKLP (Mean Molar Ratio) SD Molar Ratio n
1 F151 IA100 5 0.08 0.04 7 0.15 0.04 4
1 B21 IA 100 1 0.15 0.04 3 0.67 1
1 122 ΙΑ100 1 0.07 0.02 3 0.21 1
1 154 ΙΑ100 1 0.15 0.05 3 0.35 1
2 C63 IA 100 1 0.08 0.02 3 5.85 1
3 EE1 1.03 0.52 5 0.86 0.52 3 0.57 0.36 4
3 DD20 3.45 1.34 3 1.82 0.76 3 1.31 0.86 3
DD7 2.18 0.45 3 4.22 0.95 3 5.34 1.22 2
3 JK3 1.86 0.03 2 ND 1.44 0.03 2
4 MBK3 ND ND ND
4 NR15 ND ND ND
4 NR1 ND ND ND
5 UR29 0.60 0.12 5 IA200 3 IA300 4
6 UR11 6.99 1.61 3 19.65 14.95 3 11.09 3.13 2
7 LR4 IA100 1 IA400 1 IA400 1
7 LR6 IA100 1 IA100 1 ND
7 LR12 IA100 1 IA100 1 ND
7 LR16 IA100 1 IA100 1 ND
Antibodies were preactivating calcium me was added to HEK m 8AM) on the Hamam Data was exported ai for ligand déplétion w reported as molar ra concentration of ligan Molar Ratio for ligand SD = Standard Dévia at 200nM; 1A300 = ln< ncubated with a set concentration of ligand, usually an EC70-80 for ibilization at the Bradyklnin B1 Receptor. The antibody-ligand mixture BKR1 cells pre-loaded with a calcium sensing dye (Fluo-4AM or Fluoatsu FDSS6000 Instrument, and calcium mobilization was monitored. ï a max-min relative fluorescence of the biological response, and IC50 as calculated using sigmoidal curve fit In Graph Pad Prism V4.03. Data itio for ligand déplétion by the antibody to standardize the different d that was used for the various experiments. déplétion = [IC 50 of Antibody]/ [Ligand] Bon; ND = not determined; IA100 = Inactive at 100nM; IA200 = Inactive active at 300nM; IA400 = Inactive at400nM
Table 15: Characterization of Klnln Ligand Déplétion using Calcium Moblllzatlon ln MRC5 Fêtai Lung Fibroblaste cells
Déplétion of BK Déplétion of KD Déplétion of KLP
Famlly Antibody BK (Mean Molar Ratio) SD Molar Ratio n KD (Mean Molar Ratio) SD Molar Ratio n KLP (Mean Molar Ratio) SD Molar Ratio n
1 F151 IA100 5 0.14 0.05 5 0.15 0.02 3
1 B21 IA100 1 0.33 1 ND
1 I22 IA100 1 0.22 1 ND
1 I54 IA100 1 0.30 1 ND
2 C63 IA100 1 0.23 1 ND
3 EE1 IA300 4 1A300 4 IA150 1
3 DD20 IA600 4 1A600 5 IA150 1
DD7 7.11 3.62 3 17.37 12.11 3 4.27 1
3 JK3 IA300 2 IA300 2 ND
4 MBK3 22.11 14.10 9 3.46 2.64 6 9.45 1
4 NR15 15.26 11.51 5 4.34 2.55 5 11.18 1
4 NR1 39.31 1 42.15 1 32.58 1
5 UR29 1.15 0.86 5 0.30 0.08 2 0.41 1
6 UR11 5.41 0.80 2 25.21 4.54 2 1.53 1
7 LR4 IA100 1 IA100 1 ND
7 LR6 IA100 1 IA100 1 ND
7 LR12 IA100 1 IA100 1 ND
7 LR16 IA100 1 IA100 1 ND
Antibodies were preactivating calcium me was added to MRCS sensing dye (Fluo-4A mobîlization was moi biological response, £ Graph Pad Prism V4. standardize the diffen incubated with a set concentration of ligand, usually an EC70-80 for ibilization at the Bradykinin B2 Receptor. The antibody-ligand mixture Fêtai Lung Fibroblasts (ATCC CCL-171) pre-loaded with a calcium M or Fluo-8AM) on the Hamamatsu FDSS6000 instrument, and calcium litored. Data was exported as a max-min relative fluorescence of the ind IC50 for ligand déplétion was calculated using sigmoidal curve fit in 03. Data reported as molar ratio for ligand déplétion by the antibody to ent concentration of ligand that was used for the various experiments.
Molar Ratio for ligand déplétion = = [IC50 of Antibody]/ [Ligand]
SD s Standard Déviation; ND = not determined; IA100 = Inactive at 100nM; IA150 = Inactive at 150nM; IA300 = Inactive at 300nM; IA400 = Inactive at400nM; IA600 = Inactive at 600nM
Example 5: Engineering of F151: Humanization, Stabilization and Mutation of Unwanted Sequence Motifs
1. HUMANIZATION
The humanization protocol used has been described in PCT/US08/74381 (U S20110027266), herein incorporated by reference in its entirety. The variable light (VL) and variable heavy (VH) sequences of murine F151 were used to build a homoiogy model of anti-DAKD/KD F151 LC and 10 HC in Molecular Operating Environment (MOE; v. 2009.10; Chemical Computing Group). The following templates were used: light chain framework - 1SBS (93% identity in the framework régions), heavy chain framework - 2VXT (84% identity in the framework régions), L1 - 1LVE (93% identity), L2 - 1EEU (100% identity), L3 - 2R56 (93% identity), H1 - 1NJ9 (95% identity), H2 2VXU (76% identity) and H3 - 1HIL (49% identity). Templates were available at the RCSB Protein 15 Data Bank found on the worid wide web at rcsb.org, a website managed by Rutgers and the
University of California San Diego (Berman, H.M; Westbrook J.; Feng. Z.; Giililand, G.; Bhat, T.N.;
Weissig, H.; Shindyalov, I.N.; Boume, P.E. The Protein Data Bank, Nucieic Adds Research, 2000, 2Θ, 235-242.). The homology model was subsequently energy minimized using the standard procedures implemented in MOE. A molecular dynamics (MD) simulation of the minimized 3D homology model of the murine F151 was subsequently performed, with constraints on the protein backbone at 500 K température for 1.1 nanoseconds (ns) in Generalized Bom implidt solvent. Ten diverse conformations were extracted from this first MD run every 100 picoseconds (ps) for the last 1 ns. These diverse conformations were then each submitted to a MD simulation, with no constraints on the protein backbone and at 300 K température, for 2.3 ns. For each of the 10 MD runs, the last 2,000 snapshots, one every ps, from the MD trajectory were then used to calculate, for each murine F151 amino add, Its root mean square déviations (rmsd) compared to a reference medold position. By comparing the average rmsd on the 10 separate MD runs of a given aminoacid to the overall average rmsd of ail F151 murine amino-adds, one deddes If the amlno-add Is flexible enough, as seen during the MD to be considered as likely to interact with T-cell receptors and responslble for activation of the Immune response. 62 amino-adds were identified as flexible In the murine F151 antibody, excluding the CDR and Its Immédiate 5 A vidnity.
The motion of the 28 most flexible murine F151 amino adds, during the 20 ns (10 x 2 ns), were then compared to the motion of the corresponding flexible amino-adds of 49 human germline homology models, for each of which were run the 10 x 2 ns MD simulations. The 49 human germline models were built by systematically combining the 7 most common human germline light chains (vk1, vk2, vk3, vk4, vlambdal, vlambda2, v!ambda3) and 7 most common human germline heavy chains (vh1a, vh1b, vh2, vh3, vh4, vh5, vh6). The vk1-vh1b human germline antibody showed 0.80 4D similarity of its flexible amino-adds compared to the flexible amino-acids of the murine F151 antibody; the vk1-vh1 b germline antibody was therefore used to humanize F151 antibody focusing on the flexible amino-adds. For the pair wise amino-acid association between murine F151 vk1-vh1b amino-adds, the 2 sequences were aligned based on the optimal 3D superposition of the alpha carbons of the 2 corresponding homology models (see Figure 15 for an alignment of F151 LC and F151 HC with vk1 and vh1b, respectively).
2. STABILIZATION
Two approaches were used to Improve the stability of the antibody.
a) KNOWLEDGE-BASED APPROACH
The amino adds of the light and heavy chains with low frequency of occurrence vs. their respective canonical sequences, exduding the CDRs, were proposed to be mutated into the most frequently found amino acids (AÂGth > 0.5 kcal/mol; E. Monseilier, H. Bedoueiie. J. Mol. Biol. 362,2006, p. 580-593). This first list of consensus mutations for the light chain (LC) and heavy chain (HC) was restricted to the amino acids found in the ciosest human germline (vk1-vh1b). Suggested changes in the immédiate viclnity ofthe CDRs (5Angstroms Vernier zone, J. Mol. Biol. 224,1992, p. 487499) were removed from considération. This resulted in 5 stabilizing mutations in the LC (see Table 19) and 4 stabilizing mutations in the HC (see Table 20). Other criteria were taken into account to consider these mutations for potentially stabilizing the anti-DAKD/KD F151 antibody. These criteria were a favorable change of hydropathy at the surface or a molecular mechanics based predictedstabilization ofthe mutant Also, additional stabilizing mutationsreported tobe successful in the literature (E. Monsellier & H. Bedouelle, J. Mol. Biol., 362,2006, p. 580-593; B.J, Steipe et al. J. Mol. Biol, 1994,240,188-192) were considered (see Tables 16-22). One of these changes was incorporated as a stabilizing mutation (D89E) ln sequences HC2a, HC2b and HC2c below. Another suggested change (Q62E) was incorporated in variant HC2b.
b) 3D AND MD-BASED APPROACHES
3D and MD- based approaches hâve been previously reported (Seco J, Luque FJ, Banil X., J Med Chem. 2009 Apr 23;52(8):2363-71; Malin Jonsson et al., J. Phys. Chem. B 2003,107,5511-5518). Hydrophobie régions ofthe antibody were explicitly identified by analyzing the moleculardynamics simulation ofthe Fab in a binary solvent (20% isopropanol in water, 20 ns production simulation). Lysine mutations were then introduced in the vicinity of these régions as an attempt to prevent the aggregation. Additional analysis using a hydrophobie surface map within Schrodingeris maestro software (v. 8.5.207) was completed. Using a combination of these two techniques, 2 Lys mutations, 1 in the heavy chain and 1 ln the light chain, are suggested.
3. HUMANIZATION BY GRAFTING
Humanization using grafting grafting techniques has previously been reported (Peter T. Jones, Paul H. Dear, Jefferson Foote, Michael S. Neuberger & Greg Winter
Nature, 1986, 321,522-525). The humanization process which was used started by Identifying the ciosest human germlines to anti-DAKD/KD light and heavy chains. This is done by performing a BLAST search vs. ail the human germlines which were systematically enumerated (ail possible combinations of the V & J domains for the kappa and lambda chains; V, D and J domains for the heavy chains).
The following ciosest human germlines were identified with 83% and 62% sequence Identity to antiDAKD/KD F151 light chains (LC) and heavy chains (HC), respectively (see Figure 16). Using the internai VBASE germline, the light chain is found to be close to VDIV-B3 (-83% identity) locus and the heavy chain close to 1-08 & 1-18(-62% identity) locus ofthe VH1 sub-family. CDR régions (as defined by MOE), and Vernier régions (as defined in Foote & Winter, J. Mol. Biol., 1992,224,487499) are indicated in boldface The humanizing mutations in underiining were obtained by
A performing a pairwise comparison ofthe 2 aligned sequences, excluding the CDR & Vernier zone residuesas defined above. In anothervariantofthe humanization,onlythe CDRswere excluded in the comparison.
4. MUTATION OF UNWANTED SEQUENCE MOTIFS
The following motifs of sequences were considered: Asp-Pro (add labile bond), Asn-X-Ser/Thr (glycosylation, X=any amino-add but Pro), Asp-Gly/Ser/Thr (succinimide/iso-asp formation in flexible régions), Asn-Gly/His/Ser/Ala/Cys (exposed deamidation sites), and Met (oxidation in exposed areas). Among other criteria, the VL & VH domains of murine F151 was selected from other murine antibodies because murine F151 did not hâve exposed unwanted sequence motifs, but they are introduced in some humanized variants.
LC3a, LC3b, HC3a and HC3b each hâve potentially problematic succinimide sites that were identified. These sites were not modified in the proposed sequences as the residues invoived are potentially invoived in H-bond network (visual inspection of the homoiogy model). These positions are also found in a number of other antibody structures. Additionally, in both HC3a and HC3b, a strict humanization by grafting wouid inciude a substitution of Ser115 to Met. This Méthionine is exposed. A substitution to Leucine at this position is suggested as a humanizing mutation as it is a common residue among many close human germline sequences.
The resulting humanized sequences were blasted for sequence similarity against the International Epitope Database (IEDB) database (found on the world wide web at immuneepitope.com; version June 2009; Vita R, Zarebski L, Greenbaum JA, Emami H, Hoof I, Salimi N, Damie R, Sette A, Peters B. The Immune epitope database 2.0. Nucleic Acids Res. 2010 Jan;38(Database issue):D854-62. Epub 2009 Nov 11) to ensure that none of the sequences contain any known human B- or T-celi epitopes (sequence identity of 70% used as cut-off for the results obtained through BLAST search and considering only the results from human species).
5. ORIGINAL SEQUENCES OF MURINE F151 VARIABLE DOMAINS
CDRs are highlighted In bold and Vernier régions (as defined in Foote & Winter, J. Mol. Biol., 1992, 224,487-499) are underlined.
Light Chain (SEQ ID NO:26)
DIVHSQSPSS WYQQKPGQSP ISSVKAEDLA LAVSVGEKVTMSCKSSQSLLTSSNQKNYIA KPLIYWASTRESGVPDRFTGSGSGTDFTLT IYYCQQYTSTPWTFGGGTKLEIK
Germinality index = 83% with Z46615_1_V_X67858_1_J [VDIV-B3] »
Heavy Chain (SEQ ID NO:19) :
EIQLQQSGPELVKPGTSVKVSCKASGTSFTDTNIYWVKQS
HGKSLEWIGT roPTNGNTGTNQKFRGKATLTVDKSSSTAF
MHLSSLT SDOSAVYYCANTTRTDDHAMDTWGQGTSVTVSS
Germinality index = 62% with Z12316_1_VX97051_4_D_X97051_5_J [VH1 1-18]
6. ENGINEERED SEQUENCES
a) Background versions for the light chain (LC1, LC2a, LC2b, LC3a, and LC3b) and 5 versions of the heavy chain (HC1, HC2a, HC2b, HC3a, and HC3b) were proposed.
LC1 contains 5 humanizing mutations identified using the 4D humanization protocol. LC2a introduced an additional 5 stabilizing mutations. LC2b added 1 Lysine mutations to help prevent aggregation. LC3a contains 15 mutations derived from grafting to the closest human germline sequence and retaining the murine CDR and Vernier zone residues. LC3b contained 16 mutations derived from CDR-grafting with one additional humanizing mutation.
HC1 has 6 humanizing mutations identified by the in-house protocol. HC2a introduced 5 additional stabilizing mutations while HC2b contains 6 additional stabilizing mutations as compared to HC1. HC2c contains 1 Lys mutation, in addition to the stabilizing mutations of HC2a, to help prevent aggregation. HC3a contains 19 mutations derived from grafting to the closest human germline sequence and retaining the murine CDR and Vernier zone residues. HC3b contains 25 mutations derived from CDR grafting.
combinations in total were proposed (summarized in Table 16):
• LC1 x HC1 (mutations addressing humanization only) • LC2a x HC2a (mutations addressing humanization and stabilization) • LC2a x HC2b (mutations addressing humanization and stabilization) • LC2b x HC2c (mutations addressing humanization, stabilization and ‘anti-aggregation’) • LC3a x HC3a (mutations addressing mostiy humanization by grafting + Vernier) • LC3b x HC3b (mutations addressing humanization by grafting)
Table 16: Summary ofthe 6 LCxHC combinations proposed
(LC1) Humanlzlng (LC 2a) Humanlzlng + stabüizlng + S c JH « o W _ (H E?o si» s® (LC3a) Grafting With Vemier Réglons (LC3b) Grafting
(HC1) Humanlzlng X
(HC2a) Humanlzlng + stablllzing X
(HC2b) Humanlzlng + stabllizlng X
(HC2c) Humanlzlng +stablllzlng + “antlaggregation” X
(HC3a) grafting X
(HC3b) grafting X
Table 17: Mutations of the 5 LC variants ofthe antl-DAKD/KD F151 antibody
Light Chain Sequential numbering Light Chain Kabat Numbering (LC1) Humanlzlng mutations (LC2a) Humanlzlng + stablllzing mutations (LC2b) Humanlzlng + stablllzing mutations * anti-aggregatlon mutations (LC3a) Grafting CDRs + Vemier residues (LC3b) Grafting CDRs only
Ser5 Ser5 Thr Thr Thr Thr
Ser9 Sert) Asp Asp
Ala 12 Ala 12 Ser Ser
Val13 Val13 Ala Ala Ala
Val15 Val15 Leu Leu
Glu 17 Glu17 Asp Asp Asp
Lys 18 Lys18 Arg Arg Arg Arg Arg
Val19 Val 19 Ala Ala
Met21 Met21 Ile Ile Ile Ile
Ser22 Ser22 Asn Asn
Gln48 Gln42 Lys Lys Lys
Ser49 Ser43 Pro Pro
Pro52 Pro46 Leu
Thr69 Thr63 Ser Ser Ser Ser
Val84 Val78 Leu Leu
Lys85 Lys79 Gin Gin Gin Gin Gin
Leu89 Leu83 Lys Val Val
Ile91 Ile85 Thr Thr Val Val
Gly 106 Gly 100 Gin Gin
Leu110 Le U104 Val Val
Mutations: 5 10 11 15 16
Table 18: Mutations of the 6 HC variants ol the antl-DAKD/KD F151 antibody
Heavy Chain Séquentiel numbering Heavy Chain Kabat numbering (HC1) Humanlzing mutations (HC2a) Humanlzing +stabllizlng mutations (HC2b) Humanlzing stabilizing mutations (HC2c) humanlzing +stablllzlng mutations + antlaggregation mutations (HC3a) Graftlng CDRs + Vernier residue (HC3b) Graftlng CDRs only
Glu1 Glu1 Gin Gin Gin Gin Gin
Ile2 Ile2 Val
Gln5 Gin 5 Val Val Val Val Val Val
Pro9 Pro9 Ala Ala Ala Ala Ala
Leu 11 Leu 11 Val Val Val Val Val Val
Val12 Val12 Lys Lys Lys Lys Lys Lys
Thr16 Thr16 Ala Ala Ala Ala Ala Ala
Lys38 Lys38 Arg Arg
Ser40 Ser40 Ala Ala
His41 His41 Pro Pro Pro Pro Pro Pro
Lys43 Lys43 Gin Gin
Ser44 Ser44 Gly Gly Gly Gly Gly
Ile48 Ile48 Met
Gln62 Gln61 Glu
Lys67 Lys66 Arg Arg
Ala68 Ala67 Val
Leu70 Leu69 Met
Val72 Val71 Thr
Lys74 Lys73 Thr
*
Ser76 Ser75 Thr Thr
Phe80 Phe79 Tyr Tyr Tyr Tyr Tyr
His82 His81 Glu Glu
Ser84 Ser82A Arg Arg
Leu 86 Leu82C Lys
Thr87 Thr83 Arg Arg
Asp89 Asp85 Glu Glu Glu Glu
Asp90 Asp86 Glu Glu Glu
Ser91 Ser87 Thr Thr
Sert 15 Sert 08 Leu Leu
Mutations: 6 11 12 12 19 25
a) Engineered light chain sequences:
No potentially probiematic known T-cell or B-cell epitopes were found In ali the variants proposed.
LC1 (SEQ ID NO:27), humanizing mutations are underlined, CDRs and vemier zones are in bold:
DIVMSQSPSSLAASVGDRVTMSCKSSQSLLTSSNQKNTIA
WTQQKPGKSP KPLITWASTRESGVPDRFTGSGSGTDFTLT
ISSVQAEDLAIYYCQQTTSTPWTFGGGTKLEIK
LC2a (SEQ ID NO:28), humanizing mutations are underlined, CDRs and vemier zones are ln bold, stabilization mutations are in Italics (T at position 5, S at position 12,1 at position 21, S at position 69, T at position 91 shown below) :
DIVMWSPSSLSASVGpRVTISCKSSQSLLTSSNQKNTIA
WTQQKPGKSPKPLITWRSTIŒSGVPDRFSGSGSGTDFTLT
ISSVQAEDLA TYYCQQTTSTPWTFGGGTKLEIK
LC2b (SEQ ID NO:29) humanizing mutations are underlined, CDRs and vemier zones are In bold, stabilization mutations are ln italics (T at position 5, S at position 12,1 at position 21, S at position 69, T at position 91 shown below) and an anti-aggregation mutation Is K at position 89:
DIVMTQSPSSLSASVGDRVTISCKSSQSLLTSSNQKNTIA
WTQQKPGKSPKPLITWASTRESGVPDRFSGSGSGTDFTLT
ISSVQAEDKA TYYCQQTTSTPWTFGGGTKLEIK
LC3a (SEQ ID NO:30), grafted mutations shown ln underiine and CDRs and vernier zones shown in bold:
DIVMTQSPDSLAVSLGERATINCKSSQSLLTSSNQKNTIA WTQQKPGQPPKPLITWASTRESGVPDRFSGSGSGTDFTLT
IS SLQAEDVAVYYCQQTTSTPWTFGQGTKVEIK
LC3b (SEQ ID NO:31), grafted mutations shown in underiine and CDRs and vernier zones shown in bold:
DIVMTQSPDSLAVSLGERATINCKSSQSLLTSSNQKNTLA WTQQKPGQPPKLLIXWASTRESGVPDRFSGSGSGTDFTLT
IS SLQAEDVAVYYCQQTTSTPWTFGQGTKVEIK
Note that L at position 52 is a vernier residue that is mutated to human.
c) Engineered Heavy chain sequences
HC1 (SEQ ID NO:20), humanizing mutations are underlined, CDRs and vernier zones are In bold:
EIQLVQSGPEVKKPGASVKVSCKASGTSFTDINITWVKQS
PGKSLEWIGTFDPTNGNTGTNQKFRGKATLTVDKSSSTAF MHLSSLTSEDSAVYYCANXTRTODHAMDTWGQGTSVTVSS
HC2a (SEQ ID NO:21), humanizing mutations are underlined, CDRs and vernier zones are in bold, stabilization mutations are In italics (Q at position 1, A at position 9, G at position 44, Y at position 80 and E at position 90 shown below):
QIQLVQSGAEyKKPGASVKVSCKASGTSFTOTNITWVKQS
PGKGLEWIGTroPTNGNTGTNQKFRGKATLTVDKSSSTAY
MHLSSLTSEESAVYYCANTTRÏDDHAMDTWGQGTSVTVSS
HC2b (SEQ iD NO:22), humanizing mutations are underlined, CDRs and vernier zones are in bold, stabilization mutations are in italics (Q at position 1, A at position 9, G at position 44, E at position 62, Y at position 80 and E at position 90 shown below):
QIQLVQSGAEVKKPGASVKVSCKASGTSFTDTNITWVKQS PGKGLEWIGTFDPTNGNTGTNEKFRGKATLTVDKSSSTA Y
MHLSS LTSEESAVYYCANTTRTDDHAMDTWGQGTSVTVSS
No human epitopes were identified for sequence HC2b ln IEDB database.
HC2c (SEQ ID NO:23), humanizing mutations are underlined, CDRs and vernier zones are in bold, stabilization mutations are in italics (Q at position 1, A at position 9, G at position 44, Y at position and E at position 90 shown below) and an anti-aggregation mutation at K at position 86:
QIQLyQSGAEVKKPGASVKVSCKASGTSFTDTNITWVKQS
PGKGLEWIGTTOPTNGNTGTNQKFRGKATLTVDKSSSTAY
MHLS S KTSE ES AVYYCANTTRTDDHAMDTWGQGTSVTVSS
HC3a (SEQ ID NO:24), grafted mutations shown in underiine and CDRs and vemier zones shown in bold:
QIQLyQSGAEVKKPGASVKVSCKASGYSfTDTNIYWVRQA
PGQGLEWIGTFDPTNGNTGTNQKFRGRATLTVDKSTSTAY
MELRS LRS DDTAVYYCANYÏRTDDHAMDYWGQGTLVTVS S
LC3b (SEQ ID NO:25), grafted mutations shown in underiine and CDRs and vemier zones shown in bold:
QyQLyQSGAEVKKPGASVKVSCKASGTSFTDTNITWVRQA
PGQGLEWMGTFDPTNGNTGTNQKFRGRyTMTTDTSTSTAY MELRSLRSDDTAVYYCANTYRTDDHAMDTW GQGTLVTVSS
Note that the following Vemier Residue are mutated to human: V at position 2, M at position 48, V at position 68, M at position 70 and T at position 74.
No human epitopes were identifîed for sequence HC3b ln IEDB database.
HC3b germinality index = 83% with Z12316_1_V_J00235_1_D_U42590_1_J [1-18/DP-14J.
Table 19: Stabilizing Changes Proposed ln Light Chain
Residue Proposed Change Calculated □□Gth Accept Change
Ser-5 Thr 2.32286 Yes
Ala-12 Ser 0.75228 Yes
Met-21 Ile 0.768959 Yes
Pro-52 Leu 1.70059 No - Vemier région
Thr-69 Ser 1.10843 Yes
Lys-86 Glu 2.00115 No - changed to Gin during humanization
lle-91 Thr 1.27255 Yes
Table 20: Stabilizing Changes Proposed in Heavy Chain
Residue Proposed Change Calculated □□Gth Accept Change
Glu-1 Gin 0.562423 Yes
lle-2 Val 2.15882 No - Vemier région
Pro-9 Ala 0.505324 Yes
Thr-16 Ala 1.50552 Already changed to Ala in humanization
Val-20 Leu 2.21586 No - not in germline sequence
Ser-40 Arg 1.03643 No - not in germline sequence
His-41 Pro 1.67738 Already changed to Pro ln humanization
Ser-44 Gly 1.5068 Yes
Gln-62 Glu 0.74934 No - not in germline sequence
Arg-65 Lys 2.32314 No - not in germline sequence
Phe-80 Tyr 1.30935 Yes
His-82 Gin 2.24674 No - not in germline sequence
Asp-89 Glu 1.65409 Already changed to Glu
»
in humanization
Asn-98 Arg 3.65643 No - Vernier région
Table 21: Combinations of stablllzlng mutations evaluated
Combination* Additional changes suggested Accept Change
L1 (46->P & 48->Q) K48->Q No - K48 humanizing mutation
L2 (51->K) None - already K51 None
L3 (80->T) None - already T80 None
L4 (82->S) None - already S82 None
L5 (90->A, 91->T) None - already A90, T91 suggested above (Table 1) None
H1 (15->G) None - already G15 None
H2 (62->E, 63->K, 64->F) Q62->E, already K63 and F64 Yes - considered in HC2b
H3 (87->T, 88->S, 89->D) D89->E, already T87 and S88 Yes - potentiel sait bridges with K63 and K43
S1(L1& L5) K48->Q No - K48 humanizing mutation
S2 (H1 & H3) D89->E No (see H3)
‘Note: Sequential numbering used to refer to residues
Table 22: Potentiel Stablllzlng Mutations
Light Chain Residue* Additional changes suggested Accept Change
15->L V15->L No - V15 in Vk1 germline
96->Q None - already Q96 None
38->Y None - already Y38 None
112->l None-already 1112 None
69->S G69->S No - G69 Is in Vernier Région
21->l M21->l Already changed (see Table 19)
•Note: Sequential numbering used to refer to residues
Example 6: Characterization of Humanization Variants
Based on the In siiico modeling presented in Table 16, the variable région of the light chain (VL) and heavy chain (VH) DNA of humanized F151 were codon optimlzed for HEK293 expression and gene synthesized by GeneArt (subsidiary of Llfe Technologies). The synthesized DNA fragments were doned into the constant région of the light chain (CL) encoding vectors, pFF0362 (A. Human Kappa LC vector) at ApaLI/BsiWI sites and the constant réglons of the heavy chain (CH1, CH2 and CH3) encoding vectors, pFF0363 (B. Human lgG1 HC vector) at ApaLI/Apal sites respectiveiy.
The resulted plasmids pFF0460 containing the full sequence of LC and pFF0466 containing the fui! length of HC of humanized F151 variants were co-transfected and transiently expressed in Free Style™ 293 Expression System (Invitrogen/Life Technologies, catalog no. K9000-01).
The six humanized variants shown in Table 16 were characterized by various parameters such as binding kinetics (discussed above) as well as chemical and physicai properties such as thermostability that are routinely used in the art.
The characterization was done in two tiers. Tier I induded differential scanning calorimetry (DSC) shown in Table 24 and Figure 2. Briefly, for the DCS experiments, the antibodies were dialyzed against phosphate-buffered saline solution. Antibody concentrations were measured by UV absorbance. The antibodies were diluted to 1 mg/mL using PBS. Scans were performed using a
Calorimetry Sciences Corporation N-DSC II instrument using a 0.3268 mL capillary cell with PBS In the reference cell. The scan rate was 2’C/min and the samples were scanned from 20’C to 100° C.
Ail variants, except for HC3b/LC3b showed comparable binding affinities to the parental antibody. 10 Variant HC3a/LC3a was selected over the other variants based on other physiochemical properties such as SEC data, stability and lack of aggregation (see Tables 23-25).
Table 23. Comparison of Klnetics ofthe Humanlzed F151 Variants
HC1/LC1 HC2a/LC2a
Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M)
DAKD-b 4.16E+05 6.00E-06 1.45E-11 6.66 E+05 1.22E-05 1.83E-11
KD-b 4.24E+05 1.74E-07 3.94E-13 7.03E+05 6.12E-O6 8.71 E-12
DAKLP-b 5.00E+05 7.96E-06 1.60E-11 4.10E+05 5.67E-06 1.38E-11
KLP-b 4.81 E+05 2.67E-06 5.54E-12 6.15E+05 2.68E-05 4.34E-11
HC2b/LC2a HC2c/LC2b
Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M)
DAKD-b 4.17E+05 1.05E-05 2.57E-11 4.81E+05 4.34E-05 9.01 E-11
KD-b 3.75E+05 1.66E-06 4.72E-12 5.64E+05 9.08E-O6 1.74E-11
DAKLP-b 4.46E+05 1.30E-05 2.97E-11 9.03E+05 1.10E-05 1.21 E-11
KLP-b 4.01 E+05 2.20E-06 5.76E-12 5.16E+05 1.02E-05 1.98E-11
HC3a/LC3a HC3b/LC3b
Ka(1/Ms) Kd(1/s) KD(M) Ka(1/Ms) Kd(1/s) KD(M)
DAKD-b 5.06E+05 1.28E-05 2.53E-11 3.85E+05 5.15E-05 1.35E-10
KD-b 4.27E+05 2.95E-06 6.78E-12 2.51 E+05 3.02E-06 1.44E-11
DAKLP-b 4.65E+05 1.42E-05 3.05E-11 7.04E+04 2.76E-03 4.05E-08
KLP-b 5.02E+05 5.43E-07 1.06E-12 5.39E+05 2.72E-04 5.26E-10
For comparison: Ka(1/Ms) of mF151 was 7.84E+05 for DAKD-b, 8.30E+05 for KD-b, 1.81 E+06 for DAKLP-b, and 1.12E+06 for KLP-b
| Abbreviations: Ag=aggregation, Deg=degrada1 HC3W LC3b HC3aJ LC3a r- ï Ω o Μ N □·£ HC2b/ LC2a HC2a/ I LC2a 1 HC1/ LC1 Variant
Method Test
Ib M M O en CO CO ω M O CO <0 UV Spectroscopy (A280) Protein Conc. (mg/ml)
No Ag/Deg i No Ag/Deg No Ag/Deg No Ag/Deg No I Ag/Deg | No Ag/Deg 1D-gel (reducing & non-reduclng) Purity
NoAg z o £ NoAg Z o > ta NoAg | 3? tn a 'p P* SEC
o D 79.4(M) 71.4(m) 82.3(M) 71.8(m) Most Stable 71.0(M) 64.0(m) 82.5{m) unstable 74.6(M) 83.0(m) co -m to en Cfl o 3 2 81.6(M) 70.0{m) DSC (Tm eC) (Tm of parental F151=73°C) Stabllity
Lost potency to KLP & DAKLP nM potency at DAKD, sub nM potency at KD, comparable among 5 variants FDSS Assay Functional Potency Assay
Active at DAKD and KD comparable among 5 variants FDSS Assay without prelncubation
Decreased affinity to KLP & DAKLP Kon 10E5, koffless than or equal to 10E-6 comparable among 5 variants Blacore Binding Ligand Affinity
Table 24. Tier 1 com paris on of humanlzatlon variants
Table 25. Tler 2 comparlson of humanlzatlon variants
Thermostability Stability Intactness Confirmation (LC, HC) N-termlnal sequence confirmation
Variant 1D-gel SEC Biacore SEC LC-MS N-termlnal sequenclng
HC1/ No No 45C No LC(+1Daoff) N terminal of LC
LC1 Ag/Deg Ag/Deg slightly faster off rate than 4C Ag/Deg HC(+1Daoff) GO dominant & HC intact
HC2af No No 45C No LC(spot on) N terminal of LC
LC2a Ag/Deg Ag/Deg slightly faster off rate than 4C Ag/Deg HC(spot on) GO dominant & HC intact
HC2b/ No No 45C No LC(+1Daoff) N terminal of LC
LC2a Ag/Deg Ag/Deg slightly faster off rate than 4C Ag/Deg HC(+1Da off) GO dominant & HC Intact
HC2c/ LC2b X X X X X X
HC3a/ No No 45C No LC(-2Da off) N terminal of LC
LC3a Ag/Deg Ag/Deg slightly faster off rate than 4C Ag/Deg HC(-2Da off) GO dominant & HC intact
HC3b/ LC 3 b X X X X X X
Thermostability = Incubation at 4eC (control) and 45’C for 3 days; 1D-gel was under non-
redudng conditions; Stability reduced/deglycosylation = 2 cycles of freezeffhaw; LC-MS = reduced and
Abbreviations: Ag=aggregation, Deg=degradation, X = no data presented
Table 26. Comparison of Parental F151 and humanlzed variant F151 (HC3a/LC3a)
Parental F151
Variable Heavy Chain Variable Ught Chain
Gene GAGATCCAGCTGCAGCAGTCTGGACCTGAGCTG GTGAAGCCTGGGACTTCAGTGAAGGTGTCCTGC AAGGCTTCTGGTTACTCATTCACTGACTACAAC ATCTACTGGGTGAAACAGAGCCATGGAAAGAGC CTTGAGTGGATTGGATATTTTGATCCTTACAAT GGTAATACTGGCTACAACCAGAAGTTCAGGGGC AAGGCCACATTGACTGTTGACAAGTCCTCCAGC ACAGCCTTCATGCATCTCAGCAGCCTGACATCT GATGACTCTGCAGTCTATTACTGTGCAAACTAC TATAGGTATGACGACCATGCTATGGACTATTGG GGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:127) GACATTGTGATGTCACAGTCTCCATCCT CCCTAGCTGTGTCAGTTGGAGAGAAGGT TACTATGAGCTGCAAGTCCAGTCAGAGC CTTTTATATAGTAGCAATCAAAAGAACT ACTTGGCCTGGTACCAGCAGAAACCAGG GCAGTCTCCTAAACCGCTGATTTACTGG GCATCCACTAGGGAATCTGGGGTCCCTG ATCGCTTCACAGGCAGTGGATCTGGGAC AGATTTCACTCTCACCATCAGCAGTGTG AAGGCTGAAGACCTGGCAATTTATTACT GTCAGCAATATTATAGCTATCCGTGGAC GTTCGGTGGAGGCACCAAGCTGGAAATC AAA (SEQ ID NO:128)
Protein EIQLQQSGPELVKPGTSVKVSÇKASGYSFTDYN IYWVKQSHGKSLEWIGY FDPYNGNTGYNQKFRG DIVMSQSPSSLAVSVGEKVTMSÇKSSQSL LYSSNQKNYLAWYQQKPGQSPKPLIYWA
KATLTVDKSSSTAFMHLSSLTSDDSAVYYÇANY YRYDDHAMDYWGQGTSVTVSS STRESGVPDRFTGSGSGTDFTLTISSVK AE D LAIYYÇQQYYSYPWTFGGGT K LE IK
(SEQ ID N0:19) (SEQ ID NO:26)
Humanized F151 (HC3a/LC3a)
Gene CAGATTCAGCTGGTGCAGTCTGGCGCCGAAGTG AAGAAACCTGGCGCCAGCGTGAAGGTGTCCTGC AAGGCCAGCGGCTACAGCTTCACCGACTACAAC ATCTACTGGGTCCGACAGGCTCCAGGCCAGGGA CTGGAATGGATCGGCTACTTCGACCCCTACAAC GGCAACACCGGCTACAACCAGAAGTTCCGGGGC AGAGCCACCCTGACCGTGGACAAGAGCACCAGC ACCGCCTACATGGAACTGCGGAGCCTGAGAAGC GACGACACCGCCGTGTACTACTGCGCCAACTAC TACAGATACGACGACCACGCCATGGACTACTGG GGCCAGGGCACCCTGGTCACCGTGTCCTCT (SEQ ID NO:129) GACATCGTGATGACCCAGAGCCCCGACA GCCTGGCCGTGTCTCTGGGCGAGCGGGC CACCATCAACTGCAAGAGCAGCCAGAGC CTGCTGTACTCTAGCAACCAGAAGAACT ACCTGGCCTGGTATCAGCAGAAGCCCGG CCAGCCCCCCAAGCCCCTGATCTACTGG GCCAGCACCCGCGAGAGCGGCGTGCCCG ATAGATTTTCCGGCAGCGGCTCCGGCAC CGACTTCACCCTGACCATCAGCAGCCTG CAGGCCGAGGACGTGGCCGTGTACTACT GCCAGCAGTACTACAGCTACCCCTGGAC CTTCGGCCAGGGCACCAAGGTGGAAATC AAG (SEQ ID NO:130)
Protein 01QLVQSGAEVKKPGASVKVSCKASGYS FT DYN IYWVROAPGOGLEWIGYFDPYNGNTGYNQKFRG RATLTVDKSTSTAYMELRSLRSDDTAVYYÇANY YRYDDHAMDYWGQGTLVTVSS DIVMTQSPDSLAVSLGERATINÇKSSQS LLYSSNQKNYLAWYQQKPGQPPKPLIYW ASTRESGVPDRFSGSGSGTDFTLTISSL QAEDVAVYYCQQYYSYPWTFGQGTKVEI
(SEQ ID NO:24) K (SEQ ID NO:30)
Single underscore = CDR région; double underscore = signature amino acids for identifying CDRs
For alignment of light and heavy chains of parental F151 to humanized F151 variant (HC3a/LC3a), 5 see Figure 3.
Example 7: Crystal Structure of Humanized Antibody F151 against BRK1 Ligand Kallldan and des-arg10-Kallldln
The crystal structures of humanized F151 (HC3a/LC3a) Fab bound to kallidan or des-arg10-kallidin was determined and the molecular interactions analyzed.
Kallidin powder was purchased from Phoenix Pharmaceuticals (Cat. No. 009-37). For Fab protein génération, the DNA of heavy chain (HC) VH région from humanized F151 HC3a was cloned into 6XHis tagged CH1 vector pFF0366 (6XHis disclosed as SEQ iD NO: 137). The iight chain (LC) plasmid used herewasthesame as ofthe original F151 LC3a used in F151 humanization (see Example 5). The two plasmids were co-transfected into free style HEK293 cells for Fab expression. The Fab protein was purified using cobalt-resin, buffer exchanged to 50 mM MES pH6.0,50mM NaCi before being concentrated to about 9 mg/mL. Purified F151 Fab protein was mixed with kallidin in a molar ratio of 1:2 and set up for crystallization screening. Crystallization screening was done with a wide range of conditions. The best crystal was observed under condition B10, B12 and G10 of Hampton Research screening kit PEG/ION HT. The crystals were cryo-protected with 20% glycerol in well buffer and frozen for diffraction data collection. The X-ray diffraction data for both complexes were collected at Canadian Light Source, beamline CMCF08ID. The Rmerge for the F151-KD complex is 8.9% and l/s(l) = 20.2 , while those for the F151DAKD are 7.7% and 18.5, respectively. The F151-KD structure was solved by molecular replacement in Phaser using Fab coordinates from PDB entry 3QOS, treating the Vl-Vh and ClCh1 domains as independent units. The structure was refined in autoBuster at 2.07 A resolution in space group P2i2i2i to an Rfactor of 0.205 and an Rfree of 0.228. The F151-DAKD structure was solved using the F151-KD coordinates. The structure was refined in autoBuster at 1.86 A resolution in space group P212121 to an Rfactor of 0.232 and an Rfree of 0.238.
The électron density maps shown in Figures 4 and 5 depict the binding of kallidin (KD) and DesArg10-kallidin (DAKD) to the F151 Fab and unambiguously détermine the positions of each amino acid. For kallidin, the électron density for the extreme C-terminal residue Arg10 Is not présent. This is in agreement with the observation that DAKD, which is missing the C-terminal residue arginine (shown in Table 27 below), binds equally well to F151 as KD. The IC50 values of F151 in the neutralization FDSS cellular assay towards KD and DAKD are 0.12 nM and 0.09 nM, respectively. In both cases the électron density Is weaker towards the C-termini of the peptides. Since Phe9 in KD has slîghtiy better électron density than that in DAKD, it is possible that the presence of the additional arginine at the C-terminus of KD stabilizes the C-terminus of this peptide when binding to F151 although this arginine Itself is not stable enough to be observed by X-ray. Since the two structures are essentially identical (rms between KD and DAKD is 0.139 for C atoms and 0.328 for ail atoms), ail the following discussions are based on the F151-KD structure.
Table 27. A selected list of klnln peptides
Peptide name SEQ ID NO: Sequence 123456789 10
KD(Kallidin) 1 Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
DAKD (desArg10Kallidin) 2 Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe
KLP (rodent KD ortholog) 3 Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
BK (Bradykinin) 5 Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
KD Is bound with its N-terminus buried in the interface between Fv subunits of the light and heavy chains, as shown in Figure 6. The interface between light and heavy chains are packed with aromatic amino acids, including Tyr-L42, Tyr-L93, Tyr-L100, Trp_L102, Phe-L104 and Tyr-H35, Trp-H47, Tyr-H50, Tyr-H99, Trp-H110, stabilizing each other through stacking and hydrophobie interactions. Residues from each of the CDR’s of light and heavy chains contribute to the binding. The residues along the light and heavy chains that are involved in interactions with KD as mapped on the CDRs are shown in Figures 7 and 8. CDR H3 of the heavy chain is the longest loop and the one most frequently used in the interactions with KD, forming a side cover for KD. The loop was stabilized mostly through interactions with the other two CDRs, H1 and H2 of the heavy chain, namely, Sait bridge between Asp-H101and Arg-H52 (stabilizing H1 and H3), arene-H interaction between Tyr-H102 and Tyr-H54 (stabilizing H2 and H3), H-bond between Asp-H108 and Tyr-H35 and H-bond between His-H105 and Tyr-L55 (stabilizing H3 and L2).
Comparing the KD interacting residues among the antibodies generated, it can be seen that there is similarity among the antibodies, and some were more related in use of particular amino acids for KD-interaction than others. For example, in the iight chain F151, C63 and I22 use more similar amino acids in their CDRs to bind KD, while B21 and I54 were more similar. in the heavy chain,
F151 and C63 were surprisingly unique from each other and from B21,122 and 154. The latter three 20 appear to form a group in similarity. C63 is particularly interesting in its heavy chain, that the loop length in H2 and H3 are more different from ail others. Considering the Fab as a whole, B21 and I54 were most closely related.
ln the crystal structure, we found that KD is involved in systematic hydrogen bond and hydrophobie 25 interactions with the Fab. The N-terminus of KD is buried in the Fab and harbors more intensive interactions, while the C-terminus is essentially solvent exposed. Except for the first 4 residues (Lys-Arg-Pro-Pro) (SEQ ID NO: 132), the other residues of KD gradually extend into the bulk solvent The amidinium group of Lys1 sidechain is anchored by Glu-L61 (L· light chain) through sait bridges, while the amino terminal amino group Lys 1 forms a sait bridge with Asp-H108 (H: heavy chain). The amidinium group of Lys1 sidechain also hangs over the aromatic ring of Tyr-L55,
Invoived in cation Interactions. Such intensive interactions Invoived with Lys1 tightly anchor the amino terminus of KD in the Fab. This also accounts for the importance of Lys1 in the binding of
KD to F151. Without it (i.e. bradykinin), no détectable binding to hF151 or F151 can be measured.
Like Lys1. Arg2 interacts with the Fab through a sait bridge. The guanidium group of Arg2 interacts with the sidechain of Asp-H104. The sidechain of Arg2 is also H-bonded with the mainchain carbonyl oxygen of Arg-H101. Also, the mainchain oxygen of Pro8 is H-bonded to the sidechain of Arg-H101. Tyr-H102 Is half-way intercalated into Phe8 and Pro9, involving in hydrophobie Interactions with KD. In addition to direct Interaction, numerous water-mediated H-bonds between
KD and the Fab are also seen. It is also interesting to notice that tyrosine residues are most frequently used in the interaction compared to other amino acids; 9 out of the 16 residues marked with asterisks in Figures 7 and 8 are tyrosines. Aii the residues in F151 surrounding KD appear to play a rôle in ligand binding, except for Asn-H33, which is close to Phe6 sidechain but incompatible in polarity and lack of other important interactions. Substitution with aromatic/hydrophobic residues, such Trp or Tyr to interaction with Phe8 appears to be a quick pick If affinity maturation is considered. These two aromatic amino acids are in fact seen in other antibodies (Trp in C63, and Tyr in B21,122,154). Table 28 below provides a detailed analysis of 16 KD-interacting amino acid residues marked in Figures 7 and 8 and sets forth functional substitutions that can be made in the CDR régions that should not disrupt antigen binding.
Table 28. A list of amino acid residues found around the KD binding pocket, and their rôles In KD binding and potentlal functional substitutions (light chain residues In grey-colored cells and heavy chain residues In unshaded cells)
Residue Rôle In KD Binding or CDR Stabilization Functional Substitution
Tyr-L31 •Edge-on hydrophobie Interactions with Pro4 •Along with Tyr-L38 and Tyr-L98 form three orthogonal planes thaï surround the 90deg tum of KDat Pro4 •His to add an H-bond with amide N of Pro5 •other aromatic a.a, such as Trp, and Phe
Tyr-L38 •hydrophobie stacking with Pro4 •Along with Tyr-L31 and Tyr-L98 form three orthogonal planes that surround the 90deg tum of KD at Pro4 •His to add an H-bond withcarbonyl 0 of Arg2
Tyr-L55 •cation-ü interaction with amidinium ion of Lys1 sidechain •H-bond with His-H105; Tyr-L55 -HÎS-H105 pairing adds stabilization between L2 and H3 loops •Trp to pair with H105 mutations of Gin, Asn, Glu or Asp (maintaining H-bond); Other variants of His_H105 are Tyr and Ser. •Other aromatic a.a., such as Trp, His and Phe
GIU-L61 •Forming key sait bridges with Lys1 sidechain •Asp, Gin, ASN (Asp is seen in B21 and 154 already, Figure 7)
Tyr-L97 •H-bond with amide N of Trp-L56 •Forming pocket for Arg1 extended sidechain •Aromatic a.a. such as Phe or His (too tight space forTrp)
Tyr-L98 •Along with Tyr-L31 and Tyr-L38 form three orthogonal planes that surround the 90deg tum of KD at Pro4 •Other aromatic a.a, such as Phe, Trp or His
_ •Forming pocket surface for Pro3 •Aromatic a.a., such as Phe (better
L100 • Part of the aromatic a.a. cluster interface between L/H chains, further including Tyr-L42, Tyr-L93, Trp L102, Phe-L104 and Tyr-H35, Trp-H47, TyrH5Ô? Tyr-H99, Trp-H110 •Partial stacking with Tyr-H50 hydrophobie interactions with Pro3) •Other variants seen are Thr (in B21 and I54) and His (in I22)
TrpL102 •Part of the aromatic a.a. cluster interface between L/H chains •Stacking with Trp-H47 •Other aromatic a.a, such as Tyr (in C63 and B21), Phe and His •Other hydrophobie residue, such as L (in I22)
Asn-H33 •Close to Phe6 sidechain but incompatible in polarity, no other rôles seen either; can be a target for affinity maturation •Replace with aromatic/hydrophobic a.a., such as Trp (seen in C63) or Tyr (seen inB21,122,154)
Asp-H52 •Sait bridge with Arg-H101, stabilizing H1 and H3 ioops •Mutate as a pair with Arg-H101 to reversely charged a.a., such ArgH52/Asp-H101, or a pairof hydrophobie a.a. (Leu, Ile, Val, Met, Phe, Tyr, Trp, Ala) to form a cluster with Phe6 of KD
Tyr-H54 •Close to Pro8 but no spécifie interactions •Close to Arg-H101, but no charge interactions •Mutated to negatively charged a.a. to stabilize Arg-H101, such D or E (seen In B21,122 and I54) or N or Q, also provide H-bond with carbonyl 0 of ProB (A Lys in C63, which can be charge-reversed to Glu)
Tyr-H99 •Part of aromatic interface between H and L chains •H-bond with Asn-H33 •Tiqht space •Mutate to small aromatic a.a. except W, such as Phe and His
ArgH101 • H-bond with amide of Pro8 •supported by Asp-H 52 (sait bridge) •Mutate as a pair with Asp-H52 to reversely charged a.a., such ArgH52/Asp-H101, or a pair of hydrophobie a.a. to form a cluster with Phe6 of KD
Tyr- H102 •Half-way intercatating into Phe9 and Pro8, hydrophobie interactions with KD •Phe can be better, Trp or His may be OKtoo
AspH104 •Key residue to salt-bridge with Arg2 •Glu to maintain sait bridge with Arg2 • Bigger a.a to fil! the gap from Pro3, such as Tyr as seen tn B21,122 and I54
Asp- H108 •Key residue to sait bridge with N-term -NH3+ of KD •H-bond with Tyr-H33, stabilizing H3 loop •Conserved residue! Not in CDR •Glu
Analysis ofthe conformational epitope of kallidin (KD) or desArglO-Kallidin (DAKD) revealed that It adopts a Pro4 kink conformation. As depicted In Figure 17, a hallmark of the Pro 4 kink conformation Is a type II tight tum in the main chain polypeptide backbone of KD or DAKD at 5 Proline 4 (see Richardson JS. The anatomy and taxonomy of protein structure. Adv Protein
Chem. 1981;34:167-339, which Is Incorporated by reference herein). The Pro 4 kink conformation may further defined by ail or substantially ail of the remaining amino acids of KD (1-2 and 6-9) or
DAKD adopting repeats of a sigmoid shape which align the hydrophobie side chains in a spatially stacking mode.
Example 8: in vivo Pharmacotogy of antl-BKR14_igand Antibodies in Pain Modèle f
The examples of the présent invention illustrate the in vivo efficacy of anti-BKR1 receptor-ligand antibodies in different predinical models of acute and chronic pain according to modified procedures described in (a) Saddi GM and Abbott FV., Pain (2000), 89:53-63; (b) Chen et al.,
Molecular Pain (2010), 2:6-13 and (c) Bennett GJ and Xle YK., Pain (1988), 33:87-107.
Animais
Expérimente were carried out using adult male OF1 mice (20-30 gr) for formalin studies and adult male C57BI/6J mice (25-30 gr) for both CFA and CCI studies. The mice were kept in a controlled température room under a 12-h light-dark cycle. Food and water were provided ad libitum. For ail of 10 the experiments, mice were acclimatized to the laboratory room for at least 2 hours before testing.No randomization was performed for the studies. Expérimentera performing the behavioral tests were not blind to treatment; however they were not aware of the study hypothesis. Ail procedures hâve been approved by the Comité d'Expérimentation pour la Protection de l’Animal de Laboratoire (Animal Care and Use Committee) of sanofi-aventis recherche & développement 15 and were carried out in accordance with French législation (Decree n*87-848 -19 October 1987 and decision -19 April 1988) Implementing Européen directive 86/609/EEC.
A. Formailn-lnduced acute Inflammatory pain
The formalin test was used to measure nociceptive and inflammatory pain. Indeed, intraplantar 20 injection of formalin induces an Initial acute nociceptive behavioral response (0-12 minutes), followed by a second Inflammatory-mediated response (15-45 minutes), which is attributed to spinal cord excitability.
Formaldéhyde (37%, Sigma) was diluted In saline (v/v) to obtain a 2.5% formaldéhyde concentration (i.e. = 6.25% formalin concentration). Mice were gentiy restrained and 20 pL of this solution was Injected subcutaneously into the dorsal part of one hind paw. Behavioral responses were scored immediately after formalin injection, then at 3 minutes intervals over 45 minutes as follows: (0): normal weight bearing of the injected paw; (1): Injected paw resting lightly on floor; (2): lifting-elevation of the Injected paw; (3): licking or biting the Injected paw. Group sizes were 11-12 30 male OF1 mice.
Scores were plotted versus time and areas under the curves (AUC) were calculated from the mean scores (±SEM) for both the early (0-12 min) and the late (15-45 min) phases. Reversai of pain-like behaviors was expressed as change In AUC in %.
EE1 antibody Inhibited the pain-like behavior in the late phase of the formalin test In male OF1 mice. EE1 antibody, when administered intravenously 48 hours before intraplantar injection of formalin, showed a dose dépendent reversai of the pain-like behavior only in the late phase with a Minimal Effective Dose (MED)=2.5 mg/kg, as depicted In Figure 9. Indeed, when administered at
2.5,10 and 30 mg/kg, EE1 reversed the late phase by 35±5%, 33±5% and 45±7%, respectively, as depicted in Table 29.
ln contrast, F151 weakly Inhibits the pain-like behavior in the late phase of the formalin test when administered 48 hours before intraplantar injection of formalin. Indeed, when administered at 2.5 and 10 mg/kg, F151 reversed the late phase by 1517% and 21 ±5%, respectively, as depicted ln Table 29.
Table 29. Effect of EE1 and F151 antibodies on formalln-lnduced pain-like behavior in male 10 OF1 mice
Group Dose (mg/kg, l.v.) A.U.C. 1SEM (15-45 min) Reversai of pain-like behavior (ln %) 1SEM (15-45 min)
Isotype* control 1B7.11 (EE1) 30 63.612.9 015
EE1 2.5 10 30 41.613.4 (***) 42.912.9 (*) 36.514.3 (*) 3515 3315 4517
Isotypecontroi 1B7.11 (F151) 10 57.313 015
F151 2.5 10 48.913.8 (NS) 45.012.8 (·) 1517 2115
·, p<0.05; ***,p<0.001: Studenfs t-test versus ad NS: non significant equate control was used.
B. CFA (Complété Freund’s adjuvant)4nduced chronic Inflammatory pain
Chronic inflammation was induced under brief anesthésia (Isoflurane, 3%) by an intraplantar administration of 25 pL of Complété Freund’s Adjuvant (CFA) containing 1pg/pL heat-killed
Mycobacterium tuberculosis in minerai oil and mannide monooleate (Sigma). Group sizes were 8 male C57B1/6 mice.
EE1 antibody was administered intravenously 22 hours after intrapiantar CFA injection at 2.5 and mg/kg and mechanical and thermal hypersensitivities were assessed at Day 1 (D1), Day 4 (D4) and Day 7 (D7) post-CFA intrapiantar administration.
B1. Mechanical Hypersensitivity
Mechanical hypersensitivity was assessed by measuring the Frequency of withdrawal Response (FR, in %) following 10 applications of a 0.6 g Von Frey filament (Bioseb, France) onto the plantar surface of the Injected paw.
To Investigate the efficacy of EE1 antibody on pain-like behavior, we calculated the reversai of mechanical hypersensitivity (in %) as follows:
Percent reversais were calculated as (Mean FR-isotype-controlportto«- FR-lpslposMose) / (Mean FRisotype-controlposuoM- Mean FR-shampcswose) for each mouse.
At D1, D4 and D7 after Intrapiantar injection of CFA, a significant increase of FR to the Von Frey filaments was observed in the isotype-control 1B7.11-treated group in comparison with the naive group, demonstrating the development of mechanical hypersensitivity. EE1 antibody, when administered intravenously 22 hours after intrapiantar CFA, was able to significantly decrease this FR at the different times studied compared with that obtained in the isotype-control 1 B7.11-treated 20 group. (Figure 10).
Reversai of mechanical hypersensitivity was 41 ±8% and 22±8% at D1,36±9% and 32±9% at D4 and 27±10% and 50±9% at D7 for a 2.5 mg/kg and 30 mg/kg intravenous administration of EE1 antibody, respectively (Table 30).
»
Table 30. Effect of EE1 antibody on CFA-Induced mechanîcal hypersensftlvlty ln male
C57BI/6 mice
Group Dose (mgfkg, l.v.) Day 1 post-CFA FR (%) % effect Day 4 post-CFA FR(%) % effect Day 7 post-CFA FR (%) % effect
Naïve n.a. 41.314.4 100111 43.812.6 10018 4515 10011 3
Isotypecontrol 1B7.11 30 81.314.4 0111 78.813 018 82.512.5 017
EE1 2.5 30 6513.3 (**) 72.513.1 (*) 4118 2218 66.313.2 O 67.513.1 f) 3619 3219 72.513.7 (*) 63.813.2 (***) 27110 5019
FR: Frequency of Response (in %) 1 SEM, % effect ± SEM, n.a. not applicable *, p<0.05, **, p<0.01 and **·, p<0.001, Two-Way ANOVA with time as repeated measure followed by Dunnetf s test for factor group for each level of factor time
B2. Thermal Hypersensltlvlty
For thermal hypersensitivity, measures of Paw Withdrawal Latendes (PWL, ln seconds) in response to a radiant heat using a planter apparatus (IITC, Woodland Hills, USA) were assessed.
To Investi g a te the efficacy of EE1 antibody on pain-iike behavior, we calculated the reversai of thermal hypersensitivity (in %) as foliows:
Percent reversais were calculated as (PWLpostdose - Mean isotype-controlpoewo») / (Mean IsotypecontrolpredoM- Mean isotype-controlposwcee) for each mouse.
Thermal hypersensitivities were not different between ail groups at baseline, before intraplantar injection of CFA (data not shown).
At D1, D4 and D7 after Intraplantar Injection of CFA, a sïgnificant decrease ln paw withdrawal iatency of the Injected paw was observed in Isotype-control 1B7.11-treated group of mice, demonstrating that CFA Induced a thermal hypersensitivity (data not shown).
EE1 antibody, administered intravenously 22 hours after intraplantar CFA injection (i.e. on Day 1 post-lntraplantar CFA Injection), and was not able to Increase the Paw Withdrawal Latency at D1, whatever the dose tested (Figure 11). However, EE1 significantly increased the Paw Withdrawal Latency at D4 and this effect was also présent at D7 (Figure 11).
Reversai of thermal hypersensitivity was 41115% and 58121% at D4 and 46110% and 52117% at D7 for a 2.5 mg/kg and 30 mg/kg intravenous administration of EE1, respectively (Table 31).
«
Table 31. Effect of EE1 antibody on CFA-Induced thermal hypersensltlvity In male C57BI/6 mice
Group Dose (mg/kg, l.v.) Day 1 post-CFA PWL (sec) % effect Day 4 post-CFA PWL (sec) % effect Day 7 post-CFA PWL (sec) % effect
Isotype* control 1B7.11 30 3.310.3 n.a. 4.110.1 n.a. 3.610.3 n.a.
EE1 2.5 30 3.110.3 2.610.2 -7110 -2015 5.210.4 5.710.6 (*) 41115 58121 5.010.3 (·) 5.210.6 (·) 46110 52117
PWL: Paw withdrawal latency 1 SEM, % effect ± SEM, n.a. not applicable ·, p<0.05, Two-Way ANOVA with time as repeated measure followed by Dunnetfs test for factor group for each level of factor time
C. CCI (Chronlc Constrlctlon lnjury)4nduced neuropathlc4ike pain (Bennett’s model)
CCI model was used as a model of peripheral nerve injury. Briefly, mice were anesthetized with Isoflurane (3%), and the right sciatic nerve was exposed at mid thigh level through a small incision. Three loose ligatures of 6.0 chromic gut (Ethicon) at 1 mm space were placed around the sciatic nerve. The surgical procedure was completed by closing the muscles and skin. The day of CCI surgery was considered as Day 0. Group sizes were 6-10 male C57BI/6 mice.
EE1 antibody was administered Intravenously at 2.5 and 30 mg/kg on Day 11 post surgery and mechanical and thermal hypersensitivities were assessed on Day 12 (D12), Day 14 (D14) and Day 18 (D18) post-surgery which corresponded to Day 1 (D1). Day 3 (D3) and Day 7 (D7) posttreatment
C1. Mechanical Hypersensltlvity
Mechanical hypersensitivity was assessed by measuring hind paw withdrawal thresholds (on both injured [i.e. Ipsi] and non-injured [i.e. Contra] paws) to an increasing pressure (in g) stimulus using a Dynamic Plantar Aesthesiometer (Ugo-Basile, Italy); a steel rod was applied to the hind paws of the mice with an increasing force (5 grams in 10 seconds).
To învestigate the efficacy of EE1 antibody on pain4ike behavior, we determined the reversai of mechanical hypersensitivity as follows: percent reversais were calculated as (Ipsipostdow - Ipsipredo») / (ContrapvfoM - lpsipmfo«e) for each mouse.
Following surgery, operated mice developed a robust sensitization to mechanical stimulus on the Injured paw, whereas the non-injured paw was not affected. At Day 11, the mechanical sensitization on the Injured paw reached a plateau (data not shown).
EE1 antibody, administered intravenously on Day 11 demonstrated a slight tendency to reverse CCi-induced mechanical hypersensitivity on D12, D14 and D18 with 15.2±4.9% and 15.2±5.7% on D12,26.8±5.7% and 25.7±4.5% on D14 and 30.3±7.1% and 20.8±5.9% on D18, at 2.5 and 30 mg/kg respectiveiy (Figure 12 and Table 32).
Table 32. Effect of EE1 antibody on CCI-Induced mechanical hypersensitivity ln male
C57B1/6 mice
Group Dose (mg/kg, l.v.) Day 12 post-CCI % effect ±SEM Day 14 post-CCI % effect ± SEM Day 18 post-CCI % effect ± SEM
Isotypecontrol 1B7.11 30 0.213.0 1.814.3 18.116.6
EE1 2.5 15.214.9 26.815.7 (·*) 30.317.1
30 15.215.7 25.714.5 (*) 20.815.9
·, p<0.05, and ·*,ρ<0.01 Two-Way ANOVA with time as repeated measure followed by Dunnett’s test for factor group for each level of factor time (statistics performed on Delta ipsi values)
C2. Thermal Hypersensitivity
For thermal hypersensitivity, measures of Paw Withdrawai Latencies (in seconds) in response to a radiant heat using a plantar apparatus (IITC, Woodland Hills, USA) were assessed on the injected hind paw.
To Investigate the efficacy of EE1 antibody on paln-like behavior, we calculated the reversai of thermal hypersensitivity (in %) as follows:
Percent reversais were calculated as (Ipsîposwo»·- Mean isotype-controlposwose) / (Mean naiveposwose - Mean isotype-controlportiwe) for each mouse.
Following surgery, operated mice developed a robust sensitization to thermal stimulus on the
Injured paw, whereas the non-injured paw was not affected. At Day 11, the thermal sensitization on the injured paw reached a plateau (data not shown).
EE1 antibody, administered Intravenously on Day 11 did not significantly increase Paw Withdrawai 20 Latency of the injured paw on D12, even If a trend was observed. However, from D14, EE1 antibody significantly increased the Paw Withdrawai (Figure 13).
Reversai of thermal hypersensitivity was 41±16% and 56124% at D12 and 51116% and 98148% at D14 and 78119% and 84122% at D18, for a 2.5 mg/kg and 30 mg/kg intravenous administration of EE1 antibody, respectively (Table 33).
Table 33. Effect of EE1 antibody on CCI-Induced thermal hypersensitivity ln male C57BI/6 mice
Klnetlc évaluation
Group Dose (mg/kg, l.v.) Day 12 post-CCI PWL (sec) % effect Day 14 post-CCI PWL (sec) % effect Day 18 post-CCI PWL (sec) % effect
naïve n.a. 6.310.5 100119 6.210.4 100114 6.110.5 100117
Isotypecontrol 1B7.11 30 3.810.2 017 3.410.2 015 3.410.2 017
EE1 2.5 30 4.810.4 5.2±0.8 41116 56124 4.810.5 6.111.30 51116 98148 5.510.5 f) 5.710.8 (**) 78119 84122
PWL: Paw withdrawal latency ± SEM, % effect ± SEM, n.a. non applicable *, p<0.05, and **,p<0.01 Two-Way ANOVA with time as repeated measure followed by Dunnetfs test for factor group for each level of factor time

Claims (17)

  1. We claim:
    1. An isolated monoclonal antibody or antigen binding fragment thereof that:
    a) specifically binds to Kallidin or des-Argio-Kallidin but not to Bradykinin or des-ArgçBradyktnin;
    b) specifically binds to Kallidin or des-Argio-Kallidin with a KD of less than IxlŒ10 M;
    c) specifically binds to Kallidin or des-Argio-Kallidin with a Kotrof less than lxlO4 s1;
    d) specifically binds to Kallidin or des-Argio-Kallidin and inhibîts binding to the bradykinin Bl receptor;
    e) specifically binds to the N-termînal Lysine residue of Kallidin or des-ArglO-Kallidin;
    f) inhibîts the binding of Kallidin or des-Argio-Kallidin to a bradykinin-1 receptor; and/or
    g) specifically binds to mouse Kallidin-like peptide (KLP).
  2. 2. The antibody or antigen binding fragment thereof of claim I comprising:
    i) a heavy chain variable domain comprising an HCDR3 amino acid sequence selected from the group consisting of:
    a) SEQ ID NO: 7 [XiY Xi XjD X4HAM X5Y], wherein
    Xi isY, For H,
    X2 is R, D, A, V, L, I, M, F, Y or W,
    XjisY,F, Wor H,
    X< is D, E or Y, and,
    Xs is D or E;
    b) SEQ ID NO: 63 [XiEYDGXîYXjXîLDXj], wherein
    Xtis WorF,
    X2 is N or no amino acid;
    X3 is Y or S,
    X4 is D or P, and
    XsisForY;
    c) SEQID NO: 13;
    d) SEQ ID NO: 32;
    e) SEQ IDNO: 40;
    f) SEQ IDNO: 47; and
    g) SEQ IDNO: 55;
    ii) a heavy chain variable domain comprising an HCDR2 amino acid sequence selected from the group consisting of:
    h) SEQ ID NO: 8 [YFX1PX2NGNTGYNQKFRG], wherein
    Xi is D, R, A, V, L, I, M, F, Y or W, and
    XîisY.D, E, N, orQ;
    i) SEQ ID NO: 64 [WX1DPENGDX2X3YAPKFQG], wherein
    Xi is I, or V,
    X2 îs T, or S, and
    Xj îs G, or D;
    j) SEQ IDNO: 14
    k) SEQ IDNO: 33;
    l) SEQ IDNO: 41;
    m) SEQ IDNO: 48;and
    n) SEQ IDNO: 56;
    iii) a heavy chain variable domain comprising an HCDRI amino acid sequence selected from the group consisting of:
    o) SEQ ID NO: 9 [GYSFTDYXilY], wherein Xi is N, W or Y;
    p) SEQ ID NO: 65 [GFNIKDYYXiH], wherein Xi is L, or M;
    q) SEQ IDNO: 15;
    r) SEQ IDNO: 34;
    s) SEQ IDNO:42;
    t) SEQ IDNO:49;and
    u) SEQ IDNO: 57;
    îv) a light chain variable domain comprising an LCDR3 amino acid sequence selected from the group consisting of:
    v) SEQ ID NO: 10 [QQ Xi X2S XjP X4TJ, wherein
    Xt isY, F or H,
    X2 is Y, F, H or W,
    Xj is Y, F, T or H, and,
    X4 îs W, Y, F, H or L:
    w) SEQ ID NO: 66 [QXiXîXîSXîPXjT], wherein
    Xt isQorN,
    X2 is Y, F, D or H,
    Xj is Y, F, H or W,
    X4 is Y, F, T or H, and
    Xsis W, Y, F.Hor L;
    x) SEQ ID NO: 69 [XtQGTHFPYT], wherein Xt is L or M;
    z) SEQID NO: 16;
    aa) SEQID NO: 35;
    bb) SEQID NO: 43;
    cc) SEQIDNO:50;and dd)SEQIDNO: 58;
    vi) a light chain variable domain comprising an LCDR2 amino acid sequence selected from the group consisting of:
    ee) SEQ ID NO: 11 [WASTRXt], wherein Xt is E, D, Q or N;
    fi) SEQ ID NO: 67 [X2ASTRX2], wherein
    Xt is W or G, and
    X2 is E, D, Q or N;
    gg) SEQID NO: 17;
    hh) SEQID NO: 36;
    ti)SEQIDNO: 51;and jj) SEQ ID NO: 59;
    and/or vii) a light chain variable domain comprising an LCDRI amino acid sequence selected from the group consisting of:
    kk) SEQ ID NO: 12 [KSSQSLL XiSSNQKN X2LA], wherein
    Xi is W, H, Y or F, and
    X2 is H or Y;
    11) SEQ ID NO: 68 [KSSQSLLXiX^XjQXxNXjLA], wherein
    Xi is W, H, Y or F,
    X2 is S or G,
    X3 isNor D,
    X4 is K or R,
    Xs is H or Y.
    mm) SEQ ID NO: 70 [KSSQSLLYSNGX1TYLN],wherein XI is K or E;
    nn) SEQID NO: 18;
    00) SEQID NO: 37;
    pp) SEQ ID NO: 44;
    qq) SEQ ID NO: 52; and rr) SEQID NO: 60.
  3. 3. The antibody or antigen binding fragment thereof of claim 1 comprising:
    a) a heavy chain variable région comprising the HCDR3, HCDR2 and HCDRI région amino sequences set forth în SEQ ID NOs 13,14, and 15, respectively, and one or more amino acid substitution at positions selected from the group consisting of Hl, H5, H9, Hl I, H12, H16, H38, H40, H4I, H43, H44, H66, H75, H79, H8I, H82A, H83, H87, and H108 according to Kabat;
    b) a light chain variable région comprising the LCDR3, LCDR2 and LCDRI région amino sequences set forth in SEQ ID NOs 16,17, and 18, respectively, and one or more amino acid substitution at positions selected from the group consisting of L5, L9, LIS, L18, L19, L21, L22, L43, L63, L78, L79, L83, L85, L100 and LI04, according to Kabat;
    c) a light chain variable région comprising the LCDR3, LCDR2 and LCDRI région amino sequences set forth in SEQ ID NOs 16,17, and 18, respectively, and one or more amino » ‘ 4
  4. 7. The îsolated monoclonal antibody or antigen binding fragment thereof of claim I wherein the antibody further specifïcally binds to a conformational epitope of kallidin (KD) or desArglOKallidin (DAKD) which adopts a Pro4 kink conformation comprising a type II tight tum at Proline 4 of the KD or DAKD.
  5. 8. The antibody or antigen binding fragment of claim 7, wherein the Pro 4 kink conformation of KD or DAKD further comprises amino acid repeats of a sigmoid shape which align the hydrophobie side chains of the amino acids in a spatial! y stacking mode.
  6. 9. The antibody or antigen binding fragment thereof of claim I conjugated to a diagnostic or therapeutic agent.
  7. 10. The isolated nucleic acid encoding the amino acid sequence of the antibody, or antigen binding fragment thereof, of claim 1.
  8. 11. A recombinant expression vector comprising the nucleic acid of claim 10.
  9. 12. A host cell comprising the recombinant expression vector of claim 11.
  10. 13. A method of producing an antibody that binds specifïcally to Kallidin and des-ArgioKallidin, comprising culturing the host cell of claim 12 under conditions such that an antibody that binds specifïcally to Kallidin and des-Argio-Kallidin is produced by the host cell.
  11. 14. A pharmaceutical composition comprising the antibody, or antigen binding fragment thereof, of claim I and one or more pharmaceutically acceptable carrier(s).
  12. 15. A method for use of the antibody or antigen binding fragment thereof of claim I to manufacture a médicament for the treatment of a Kallidin or des-Argio-Kallidin-associated disease or disorder.
  13. 16. The method of claim 15, wherein the disease or disorder is chronic pain.
    <
  14. 17. A method of generating the antibody or antigen binding fragment thereofof claim l that specifically binds to des-Arg?- Bradykinin and des-Argio-Kallidin-like peptide comprising: immunizing an animal with an immunogen comprising a peptide, wherein the peptide consists of the amino acid sequence set forth in SEQ ID No. 11, and wherein the amino terminal arginine of the peptide is indirectly coupled to a carrier moiety through a linker moiety, such that an antibody that specifically binds to des-Argç- Bradykinin, des-Argio-Kallîdin and des-ArgioKallidîn-Iike peptide is produced by the immune System of the animal.
  15. 18. The method of claim 17, further comprising isolating from the animal, the antibody, a nucleic isolating encoding the antibody, or an immune cell expressing the antibody.
  16. 19. The method of claim 17, wherein the carrier moiety is a protein.
  17. 20. The method of claim 17, wherein the linker moiety comprises [Gly-Gly-Gly]n, wherein n isat least I.
    1/56
    543895_SA9-029PC_Seq_List.txt
OA1201400447 2012-03-28 2013-03-15 Antibodies to bradykinin B1 receptor ligands OA17139A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US61/616845 2012-03-28
FR1350953 2013-02-04

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Publication Number Publication Date
OA17139A true OA17139A (en) 2016-03-28

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